Fc RECEPTOR-BINDING POLYPEPTIDES WITH MODIFIED EFFECTOR FUNCTIONS

ABSTRACT

Disclosed are processes for producing a variant polypeptide (e.g. antibodies) having modified binding characteristics for human Fc gamma receptor IIA (CD32A) leading to increased inhibition of proinflammatory mediators while retaining binding to a target antigen via its Fv portion, which processes comprise altering the polypeptides by substitution of at least two amino acid residues at EU position 325, 326 or 328 of a human IgG CH2 region for a sequence selected from SAAF, SKAF, NAAF and NKAF. The polypeptides that can be generated according to the methods of the invention are highly variable, and they can include antibodies and fusion proteins that contain an Fc region or a biologically active portion thereof.

RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/043,071, filed Oct. 1, 2013, now issued as U.S. Pat. No.9,688,755, which is a continuation of U.S. patent application Ser. No.12/152,395, filed May 14, 2008, now issued as U.S. Pat. No. 8,546,539,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/930,276 filed May 14, 2007, the contents of each of which are hereinincorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “NOVI013C02US_SeqList”, which wascreated on Jun. 23, 2017 and is 66.9 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

This invention relates to molecules, particularly polypeptides, moreparticularly immunoglobulins (e.g. antibodies) that include a variant Fcregion wherein said variant Fc region includes at least one amino acidmodification relative to a wild-type Fc region, where variant Fc regionhas modified binding characteristics for human Fc gamma receptor IIA(CD32A) leading to prevention of proinflammatory mediators release (e.g.TNF-alpha, Interleukin (IL)-1, IL-6, IL-8 and chemokines). Thisinvention also relates to molecules, particularly polypeptides, moreparticularly immunoglobulins (e.g. antibodies) that include a variantCDR3 region, wherein the variant CDR3 region includes at least one aminoacid modified relative to a wild-type CDR3 region. This invention alsogenerally relates to methods of producing such molecules, a process formodifying an effector function and methods of using such alteredantibodies as therapeutic and diagnostic agents.

BACKGROUND OF THE INVENTION

Antibodies, or immunoglobulins, comprise two heavy chains linkedtogether by disulphide bonds and two light chains, each light chainbeing linked to a respective heavy chain by disulphide bonds. Thegeneral structure of an antibody of class IgG (i.e. an immunoglobulin(Ig) of class gamma (G) is shown schematically in FIG. 1A.

Each heavy chain has at one end a variable domain followed by a numberof constant domains. Each light chain has a variable domain at one endand a constant domain at its other end, the light chain variable domainbeing aligned with the variable domain of the heavy chain and the lightchain constant domain being aligned with the first constant domain ofthe heavy chain.

Antigen binds to antibodies via an antigen binding site in the variabledomains of each pair of light and heavy chains. Other molecules, knownas effector molecules, bind to other sites in the remainder of themolecule, i.e. other than the antigen binding sites, and this portion ofantibody will be referred to as “the constant portion” of an antibody,such sites being located particularly in the Fc region constituted bythe portions of the heavy chains extending beyond the ends of the lightchains.

The constant portion of antibodies specifically interact with receptorson “effector” cells. For example, the Fc region mediates effectorfunctions that have been divided into two categories. In the first arethe functions that occur independently of antigen binding; thesefunctions due to the major histocompatibility complex class I-relatedreceptor FcRn confer IgGs persistence in the circulation and the abilityto be transferred across cellular barriers by transcytosis (see Ghetie Vand Ward S). In the second are the functions that operate after anantibody binds an antigen; these functions involve the participation ofthe complement cascade or Fc receptor (FcR) bearing cells.

FcRs are defined by their specificity for immunoglobulin isotypes. Forexample Fc receptors for IgG antibodies are referred to as FcγR. FcRsare specialized cell surface receptors on hematopoietic cells thatmediate both the removal of antibody-coated pathogens by phagocytosis ofimmune complexes, and the lysis of erythrocytes and various othercellular targets (e.g. tumor cells) coated with the correspondingantibody.

The FcγRs play a critical role in either abrogating or enhancing immunerecruitment. Currently, three classes of FcγRs are distinguished oncells of the immune system: the high-affinity receptor Fc RI (CD64),capable of binding monomeric IgG; and the low-affinity receptors FcγRII(CD32), and FcγRIII (CD16), which interact preferentially with complexedIgG. Furthermore, the FcγRII and FcγRIII classes comprise both “A” and“B” forms (Gessner-JE et al. Ann Heamatol, 1998, The IgG Fc receptorfamily, 76: 231-248).

FcγRII proteins are 40 KDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes. The gene for the FcγRIIa receptorcontains either G or A in codon 131, resulting in either arginine (CGT)or histidine (CAT), respectively, in the second extracellular domain.This change alters the ability of the receptor to bind IgG. Cells withFcγRIIA His-131, the A/A genotype, bind human IgG2 with considerablyhigher affinity than those with Arg at position 131; conversely, cellswith Arg-131, the G/G genotype, bind murine IgG1 with considerablyhigher affinity than those with His at position 131 (Salmon et al.,1992, J. Clin. Invest. 89:1274-1281). Originally, studies usingmonocytes interaction with an anti-CD3 antibody of the murine IgG1subclass as a trigger for T-cell proliferation classified individualsphenotypically as low responders of high responders (Tax et al., 1983,Nature:304: 445-447). It is now known that high responder cells in thisassay have the G/G or A/G genotype while low-responder cells have theA/A genotype. The FcγRIIa 131 R/R genotype is a risk factor forsusceptibility to some infectious and autoimmune diseases (Van der PolW. L. and Van de Winkel J. G. J, 1998, Immunogenetics 48:222-232).

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogeneous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the B isoform initiates inhibitorysignal, e.g., inhibiting B-cell activation.

Monoclonal antibodies (mAbs) have now been used to treat disease inhuman patients. Although some mAbs may function effectively withoututilizing antibody effector functions (e.g. neutralizing antibodies), inmany cases it may be desirable to engineer the Fc portion of theantibody to recruit the immune system to elicit an immune response.There exists a need in the art to produce antibodies that include avariant Fc region having increased potency while retaining the abilityto bind to a given target. Accordingly, there exists a need to producealtered IgG antibodies that elicit a modified Fc receptor activity whileretaining binding to an antigen as compared to an unaltered antibody.

SUMMARY OF THE INVENTION

The altered polypeptides described herein include at least an FcγRbinding portion of an Fc region of an immunoglobulin polypeptide. Thealtered antibodies of the invention also include an altered antibodyhaving a variant CDR3 region in which at least one amino acid residue inthe CDR3 region of the antibody has been modified. The alteredantibodies and altered polypeptide of the invention also includepolypeptides that include at least an FcγR binding portion of an Fcregion of an immunoglobulin polypeptide and a variant CDR3 region. Thealtered antibodies and altered polypeptide of the invention also includepolypeptides that include at least a variant Fc region of animmunoglobulin polypeptide and a variant CDR3 region.

The altered polypeptides described herein include antibodies thatinclude at least one specific amino acid substitution within forexample, an Fc region or an FcR binding fragment thereof (e.g.,polypeptide having amino acid substitutions within an IgG constantdomain) such that the modified antibody elicits alterations inantigen-dependent effector function while retaining binding to antigenas compared to an unaltered antibody. For example, the alteredantibodies elicit the prevention of proinflammatory mediator release. Ina preferred embodiment, the altered antibodies are human and of the IgGisotype. For example, the altered antibodies are human IgG1, IgG2, IgG3or IgG4 isotype. The altered antibodies described herein have anincreased potency as compared to an unaltered antibody.

The altered antibodies of the invention include an altered antibody inwhich at least one amino acid residue in the constant region of the Fcportion of the antibody has been modified. For example, at least oneamino acid in the CH2 domain of the Fc portion has been replaced by adifferent residue, i.e., an amino acid substitution. In the alteredantibodies described herein, one or more of the amino acid residues thatcorrespond to residues 325, 326 and 328 is substituted with a differentresidue as compared to an unaltered antibody. The numbering of theresidues in the gamma heavy chain is that of the EU index (see Edelman,G. M. et al., 1969; Kabat, E, A., T. T. Wu, H. M. Perry, K. S.Gottesman, and C. Foeller., 1991. Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed. U.S. Dept. of Health and Human Services, Bethesda,Md., NIH Publication n. 91-3242).

These altered antibodies with a mutated Fc portion elicit modifiedeffector functions, e.g., a modified Fc receptor activity, as comparedto an unaltered antibody. For example, the human Fc receptor is CD32A.In some embodiments, the altered antibodies elicit increased inhibitionof proinflammatory mediator release following ligation to CD32A ascompared to an unaltered antibody. Thus, the altered antibodiesdescribed herein elicit a modified Fc receptor activity, such asincreasing the inhibition of proinflammatory mediators release, whileretaining the ability to bind a target antigen. In some embodiments, thealtered antibody is a neutralizing antibody, wherein the alteredantibody elicits a modified Fc receptor activity, while retaining theability to neutralize one or more biological activities of a targetantigen via Fv binding.

In embodiments where the altered antibody is a human IgG1 isotype, thealtered antibodies include the amino acid substitution at EU amino acidposition 328 alone or together with EU amino acid positions 325 and 326of the mouse IgG1 gamma heavy chain constant region as compared tounaltered antibody. In one embodiment, EU amino acid position 328 of thegamma heavy chain constant region is substituted with a non-polar aminoacid, such as alanine, cysteine, leucine, isoleucine, valine, glycine,phenylalanine, proline, tryptophan and tyrosine. Most preferably, EUamino acid position 328 of the gamma heavy chain constant region issubstituted with phenyalanine. In one embodiment, EU amino acid position325 of the gamma heavy chain constant region is substituted with a polaramino acid such as arginine, asparagine, glutamine, glutamic acid,histidine, lysine, serine or threonine. Most preferably, EU amino acidposition 325 of the gamma heavy chain constant region is substitutedwith serine. In one embodiment, EU amino acid position 326 of the gammaheavy chain constant region is substituted with a non-polar amino acid,such as alanine, cysteine, leucine, isoleucine, valine, glycine,phenylalanine, proline, tryptophan and tyrosine. Most preferably, EUamino acid position 326 of the gamma heavy chain constant region issubstituted with alanine. In some embodiments, the altered antibodiescontain EU amino acid position 328 with one or two amino acidsubstitutions within the human IgG1 gamma heavy chain constant region,wherein the substitutions occur at one or two amino acid residuesselected from EU amino acid positions 325 and 326. In one embodiment,the altered human IgG1 antibody contains amino acid substitutions at EUpositions 326 and 328. For example, the residue 326 of the human IgG1gamma heavy chain constant region is substituted with alanine and theresidue 328 of the human IgG1 gamma heavy chain constant region issubstituted with phenylalanine. In some embodiments, EU positions 325 to328 of the gamma heavy chain constant region of the altered human IgG1antibody consist of a sequence selected from SAAF (SEQ ID NO: 86), SKAF(SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF (SEQ ID NO: 89).

In embodiments where the altered antibody is a human IgG2 isotype, thealtered antibodies include the amino acid substitution at EU amino acidposition 328 alone or together with EU amino acid positions 325 and 326as compared to unaltered antibody. In one embodiment, EU amino acidposition 328 of the gamma heavy chain constant region is substitutedwith a non-polar amino acid, such as alanine, cysteine, leucine,isoleucine, valine, glycine, phenylalanine, proline, tryptophan andtyrosine. Most preferably, EU amino acid position 328 of the gamma heavychain constant region is substituted with phenyalanine. In oneembodiment, EU amino acid position 325 of the gamma heavy chain constantregion is substituted with a polar amino acid such as arginine,asparagine, glutamine, glutamic acid, histidine, lysine, serine orthreonine. Most preferably, EU amino acid position 325 of the gammaheavy chain constant region is substituted with serine. In oneembodiment, EU amino acid position 326 of the gamma heavy chain constantregion is substituted with a non-polar amino acid, such as alanine,cysteine, leucine, isoleucine, valine, glycine, phenylalanine, proline,tryptophan and tyrosine. Most preferably, EU amino acid position 326 ofthe gamma heavy chain constant region is substituted with alanine. Insome embodiments, the altered antibodies contain EU amino acid position328 with one or two amino acid substitutions within the human IgG2 gammaheavy chain constant region, wherein the substitutions occur at one ortwo amino acid residues selected from EU amino acid positions 325 and326. In one embodiment, the altered human IgG2 antibody contains aminoacid substitutions at EU positions 326 and 328. For example, the residue326 of the human IgG2 gamma heavy chain constant region is substitutedwith alanine and the residue 328 of the human IgG2 gamma heavy chainconstant region is substituted with phenylalanine. In some embodiments,EU positions 325 to 328 of the gamma heavy chain constant region of thealtered human IgG2 antibody consist of a sequence selected from SAAF(SEQ ID NO: 86), SKAF (SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF(SEQ ID NO: 89).

In embodiments where the altered antibody is a human IgG3 isotype, thealtered antibodies include the amino acid substitution at EU amino acidposition 328 alone or together with EU amino acid positions 325 and 326as compared to unaltered antibody. In one embodiment, EU amino acidposition 328 of the gamma heavy chain constant region is substitutedwith a non-polar amino acid, such as alanine, cysteine, leucine,isoleucine, valine, glycine, phenylalanine, proline, tryptophan andtyrosine. Most preferably, EU amino acid position 328 of the gamma heavychain constant region is substituted with phenyalanine. In oneembodiment, EU amino acid position 325 of the gamma heavy chain constantregion is substituted with a polar amino acid such as arginine,asparagine, glutamine, glutamic acid, histidine, lysine, serine orthreonine. Most preferably, EU amino acid position 325 of the gammaheavy chain constant region is substituted with serine. In oneembodiment, EU amino acid position 326 of the gamma heavy chain constantregion is substituted with a non-polar amino acid, such as alanine,cysteine, leucine, isoleucine, valine, glycine, phenylalanine, proline,tryptophan and tyrosine. Most preferably, EU amino acid position 326 ofthe gamma heavy chain constant region is substituted with alanine. Insome embodiments, the altered antibodies contain EU amino acid position328 with one or two amino acid substitutions within the human IgG3 gammaheavy chain constant region, wherein the substitutions occur at one ortwo amino acid residues selected from EU amino acid positions 325 and326. In one embodiment, the altered human IgG3 antibody contains aminoacid substitutions at EU positions 326 and 328. For example, the residue326 of the human IgG3 gamma heavy chain constant region is substitutedwith alanine and the residue 328 of the human IgG3 gamma heavy chainconstant region is substituted with phenylalanine. In some embodiments,EU positions 325 to 328 of the gamma heavy chain constant region of thealtered human IgG3 antibody consist of a sequence selected from SAAF(SEQ ID NO: 86), SKAF (SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF(SEQ ID NO: 89).

In embodiments where the altered antibody is a human IgG4 isotype, thealtered antibodies include the amino acid substitution at EU amino acidposition 328 alone or together with EU amino acid positions 325 and 326as compared to unaltered antibody. In one embodiment, EU amino acidposition 328 of the gamma heavy chain constant region is substitutedwith a non-polar amino acid, such as alanine, cysteine, leucine,isoleucine, valine, glycine, phenylalanine, proline, tryptophan andtyrosine. Most preferably, EU amino acid position 328 of the gamma heavychain constant region is substituted with phenyalanine. In oneembodiment, EU amino acid position 325 of the gamma heavy chain constantregion is substituted with a polar amino acid such as arginine,asparagine, glutamine, glutamic acid, histidine, lysine, serine orthreonine. Most preferably, EU amino acid position 325 of the gammaheavy chain constant region is substituted with serine. In oneembodiment, EU amino acid position 326 of the gamma heavy chain constantregion is substituted with a non-polar amino acid, such as alanine,cysteine, leucine, isoleucine, valine, glycine, phenylalanine, proline,tryptophan and tyrosine. Most preferably, EU amino acid position 326 ofthe gamma heavy chain constant region is substituted with alanine. Insome embodiments, the altered antibodies contain EU amino acid position328 with one or two amino acid substitutions within the human IgG4 gammaheavy chain constant region, wherein the substitutions occur at one ortwo amino acid residues selected from EU amino acid positions 325 and326. In one embodiment, the altered human IgG4 antibody contains aminoacid substitutions at EU positions 326 and 328. For example, the residue326 of the human IgG4 gamma heavy chain constant region is substitutedwith alanine and the residue 328 of the human IgG4 gamma heavy chainconstant region is substituted with phenylalanine. In some embodiments,EU positions 325 to 328 of the gamma heavy chain constant region of thealtered human IgG4 antibody consist of a sequence selected from SAAF(SEQ ID NO: 86), SKAF (SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF(SEQ ID NO: 89).

The altered antibodies of the invention also include an altered antibodyhaving a variant CDR3 region in which at least one amino acid residue inthe CDR3 region of the antibody has been modified. The alteredantibodies and altered polypeptide of the invention also includepolypeptides that include at least an FcγR binding portion of an Fcregion of an immunoglobulin polypeptide and a variant CDR3 region. Thealtered polypeptides described herein include antibodies that include avariant CDR3 region and at least one specific amino acid substitutionwithin for example, an Fc region or an FcR binding fragment thereof(e.g., polypeptide having amino acid substitutions within an IgGconstant domain) such that the modified antibody elicits alterations inantigen-dependent effector function while retaining binding to antigenas compared to an unaltered antibody.

The variant CDR3 regions include the variant VH CDR3 regions shown inExample 4: KDPSDAFPY (SEQ ID NO: 80) and KDPSEGFPY (SEQ ID NO: 81). Thevariant CDR3 regions include the variant VL CDR3 regions shown inExample 4: QNSHSFPLT (SEQ ID NO: 82); QQGHSFPLT (SEQ ID NO: 83);QNSSSFPLT (SEQ ID NO: 84); and QQSHSFPLT (SEQ ID NO: 85).

In some embodiments, the altered antibodies include both a variant Fcregion and a variant CDR3 region. In some embodiments, the alteredantibodies include both a variant Fc region shown in Example 3 and avariant CDR3 region shown in Example 4, e.g., SEQ ID NOs: 80-85. In someembodiments, the altered antibodies include both a variant CH2 domain inthe Fc region and a variant CDR3 region. In some embodiments, thealtered antibodies include both a variant CH2 domain in the Fc regionshown in Example 3 and a variant CDR3 region shown in Example 4, e.g.,SEQ ID NOs: 80-85. In some embodiments, the altered antibodies includeboth a variant CH2 domain in the Fc region that is mutated at one ormore of the residues that correspond to residues 325, 326 and/or 328(using the numbering of the residues in the gamma heavy chain as in theEU index, Edelman, et al.) and a variant CDR3 region. In someembodiments, the altered antibodies include both a variant CH2 domain inthe Fc region that is mutated at one or more of the residues thatcorrespond to residues 325, 326 and/or 328 (using the numbering of theresidues in the gamma heavy chain as in the EU index, Edelman, et al.)shown in Example 3 and a variant CDR3 region shown in Example 4, e.g.,SEQ ID NOs: 80-85.

The altered polypeptides and antibodies of the invention also includeantibodies that include a heavy chain variable amino acid sequence thatis at least 90%, 92%, 95%, 97%, 98%, 99% or more identical the aminoacid sequence of SEQ ID NO: 2, 12, 22, 32, 45, 46, 49, 51, 52, 66, or68, and/or a light chain variable amino acid that is at least 90%, 92%,95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQ IDNO: 7, 17, 27, 37, 47, 48, 50, 53, 71, 73, 75 or 77.

The altered polypeptides and antibodies of the invention also includepolypeptides that include at least an FcγR binding portion of an Fcregion of an immunoglobulin polypeptide, and include a heavy chainvariable amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%,99% or more identical the amino acid sequence of SEQ ID NO: 2, 12, 22,32, 45, 46, 49, 51, 52, 66, or 68, and/or a light chain variable aminoacid that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical theamino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 48, 50, 53, 71, 73,75 or 77. In some embodiments, the altered antibodies include both avariant CH2 domain in the Fc region shown in Example 3. In someembodiments, the altered antibodies include both a variant CH2 domain inthe Fc region that is mutated at one or more of the residues thatcorrespond to residues 325, 326 and/or 328 (using the numbering of theresidues in the gamma heavy chain as in the EU index, Edelman, et al.).

The altered polypeptides and antibodies of the invention also includepolypeptides and antibodies that include a variant Fc region, andinclude a heavy chain variable amino acid sequence that is at least 90%,92%, 95%, 97%, 98%, 99% or more identical the amino acid sequence of SEQID NO: 2, 12, 22, 32, 45, 46, 49, 51, 52, 66, or 68, and/or a lightchain variable amino acid that is at least 90%, 92%, 95%, 97%, 98%, 99%or more identical the amino acid sequence of SEQ ID NO: 7, 17, 27, 37,47, 48, 50, 53, 71, 73, 75 or 77. In some embodiments, the alteredantibodies include both a variant CH2 domain in the Fc region shown inExample 3. In some embodiments, the altered antibodies include both avariant CH2 domain in the Fc region that is mutated at one or more ofthe residues that correspond to residues 325, 326 and/or 328 (using thenumbering of the residues in the gamma heavy chain as in the EU index,Edelman, et al.).

The altered polypeptides and antibodies of the invention also includepolypeptides and antibodies that include a variant CH2 domain in the Fcregion, and include a heavy chain variable amino acid sequence that isat least 90%, 92%, 95%, 97%, 98%, 99% or more identical the amino acidsequence of SEQ ID NO: 2, 12, 22, 32, 45, 46, 49, 51, 52, 66, or 68,and/or a light chain variable amino acid that is at least 90%, 92%, 95%,97%, 98%, 99% or more identical the amino acid sequence of SEQ ID NO: 7,17, 27, 37, 47, 48, 50, 53, 71, 73, 75 or 77. In some embodiments, thealtered antibodies include both a variant CH2 domain in the Fc regionshown in Example 3. In some embodiments, the altered antibodies includeboth a variant CH2 domain in the Fc region that is mutated at one ormore of the residues that correspond to residues 325, 326 and/or 328(using the numbering of the residues in the gamma heavy chain as in theEU index, Edelman, et al.).

The altered antibodies and polypeptides of the invention also includepolypeptides and antibodies that include three heavy chaincomplementarity determining regions (CDRs) having an amino acid sequenceat least 90%, 92%, 95%, 97%, 98%, 99% or more identical to each of: (i)a VH CDR1 sequence selected from the group consisting of SEQ ID NOs: 3,13, 23, and 33; (ii) a VH CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 4, 14, 24, and 34; (iii) a VH CDR3 sequenceselected from the group consisting of SEQ ID NOs: 5, 15, 25, 35, 80 and81; and/or a light chain with three CDR that include an amino acidsequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to eachof (iv) a VL CDR1 sequence selected from the group consisting of SEQ IDNOs: 8, 18, 28, and 38; (v) a VL CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 9, 19, 29 and 39; and (vi) a VL CDR3 sequenceselected from the group consisting of SEQ ID NOs: 10, 20, 30, 40 82, 83,84 and 85.

The invention also provides methods of targeting human CD32A by amonoclonal antibody in which at least EU amino acid position 328 of thegamma heavy chain constant region together with one or two of the aminoacid residues that correspond to EU positions 325 and 326 of the heavygamma chain constant region are substituted with the corresponding EUamino acid residue of mouse IgG1 at the same position which is differentfrom the corresponding amino acid residue in an unaltered antibody, suchthat the antibody elicits increased inhibition of proinflammatorymediators release upon binding to human CD32A while retaining binding toantigen as compared to an unaltered antibody. In some embodiments, thealtered antibody further includes a variant VH CDR3 regions shown inExample 4: KDPSDAFPY (SEQ ID NO: 80) and KDPSEGFPY (SEQ ID NO: 81)and/or a variant CDR3 region shown in Example 4: QNSHSFPLT (SEQ ID NO:82); QQGHSFPLT (SEQ ID NO: 83); QNSSSFPLT (SEQ ID NO: 84); and QQSHSFPLT(SEQ ID NO: 85).

In some embodiments, the amino acid residue that corresponds to EUposition 325 of the gamma heavy chain constant region is substitutedwith serine. In some embodiments, the amino acid residue thatcorresponds to EU position 326 of gamma heavy chain constant region issubstituted with alanine. In some embodiments, the amino acid residuethat corresponds to EU position 328 of the gamma heavy chain constantregion is substituted with phenylalanine. In some embodiments, thealtered antibody further includes a variant VH CDR3 regions shown inExample 4: KDPSDAFPY (SEQ ID NO: 80) and KDPSEGFPY (SEQ ID NO: 81)and/or a variant CDR3 region shown in Example 4: QNSHSFPLT (SEQ ID NO:82); QQGHSFPLT (SEQ ID NO: 83); QNSSSFPLT (SEQ ID NO: 84); and QQSHSFPLT(SEQ ID NO: 85).

In some embodiments, the altered antibody binds to a target selectedfrom a toll-like receptor (TLR), MD2 accessory protein and CD14. Forexample, the altered antibody binds to soluble TLR4, the TLR4/MD2complex, or both soluble TLR4 and the TLR4/MD2 complex. In someembodiments, the altered antibody binds to TLR2. For example, theantibodies are capable of blocking, e.g., neutralizing, LPS-inducedpro-inflammatory cytokine production.

In some embodiments, the altered antibody is a human IgG1 isotypeantibody that includes at least modification of amino acid residue at EUposition 328 possibly with at least one amino acid residue of the gammaheavy chain constant region selected from amino acid residues 325 and326 wherein the altered antibody elicits a modified Fc effector activityupon binding to human CD32A while retaining binding to antigen ascompared to an unaltered antibody, and wherein the antibody includes (a)a V_(H) CDR1 region comprising the amino acid sequence of SEQ ID NO: 3,13, 23 or 33; (b) a _(H) CDR2 region comprising the amino acid sequenceof SEQ ID NO: 4, 14, 24 or 34; (c) a V_(H) CDR3 region comprising theamino acid sequence of SEQ ID NO: 5, 15, 25, 35, 80 or 81; (d) a V_(L)CDR1 region comprising the amino acid sequence of SEQ ID NO: 8, 18, 28or 38; (e) a V_(L) CDR2 region comprising the amino acid sequence of SEQID NO: 9, 19, 29 or 39; and (f) a V_(L) CDR3 region comprising the aminoacid sequence of SEQ ID NO: 10, 20, 30, 40, 82, 83, 84, or 85, whereinthe antibody binds soluble TLR4, MD2, the TLR4/MD2 complex or bothsoluble TLR4 and the TLR4/MD2 complex. In one embodiment, EU position325 of the gamma heavy chain constant region is substituted with serine.In one embodiment, EU position 326 of the gamma heavy chain constantregion is substituted with alanine. In one embodiment, EU position 328of the gamma heavy chain constant region is substituted withphenylalanine.

In some embodiments, the altered antibodies include a gamma heavy chainconstant region having two or more substitutions with an amino acidresidue that is different from the corresponding amino acid residue inan unaltered antibody, wherein the substitutions occur at EU position328 and one or two amino acid residues selected from residues 325 and326 of the gamma heavy chain constant region. In one embodiment, thesubstitutions are at residues 326 and 328. For example, EU position 326of the heavy chain constant region is substituted with alanine, and EUposition 328 of the heavy chain constant region is substituted withphenylalanine. In some embodiments, the altered antibodies contain aheavy chain constant region in which EU position 325-328 of the gammaheavy chain constant region consist of a sequence selected from SAAF(SEQ ID NO: 86), SKAF (SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF(SEQ ID NO: 89). In one embodiment, the V_(H) CDR1 region of the alteredhuman IgG1 antibody includes the amino acid sequence of SEQ ID NO: 23,the V_(H) CDR2 region includes the amino acid sequence of SEQ ID NO: 24,the V_(H) CDR3 region includes the amino acid sequence of SEQ ID NO: 25,the V_(L) CDR1 region includes the amino acid sequence of SEQ ID NO: 28,the V_(L) CDR2 region includes the amino acid sequence of SEQ ID NO: 29,and the V_(L) CDR3 region includes the amino acid sequence of SEQ ID NO:30, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 or SEQ ID NO:85.

In some embodiments, the altered antibodies are altered versions of theantibodies referred to herein as 16G7, mu16G7, 7E3, mu7E3, 15C1, mu15C1,18H10 and mu18H10. Modified versions of these antibodies, whichrecognize the TLR4/MD2 complex, elicit a modified, e.g., inhibitoryhuman CD32A activity and inhibit LPS-induced pro-inflammatory cytokineproduction at least two-fold, five-fold, 10-fold, 20-fold, 50-fold,75-fold, or 100-fold more than the commercially available, anti-TLR4non-blocking monoclonal antibody HTA125.

In some embodiments, the altered antibodies are modified versions ofantibodies that recognize CD14, such as the anti-CD14 monoclonalantibody known as 28C5 (see e.g., U.S. Pat. No. 6,444,206, herebyincorporated by reference in its entirety), and altered versions ofantibodies that recognize TLR2, including, e.g., the anti-TLR2monoclonal antibody known as T2.5 (see e.g., WO 2005/028509, herebyincorporated by reference in its entirety).

The invention also provides isolated polypeptides that include a gamma 1Fc (γ1Fc) region, wherein amino acid residues at EU positions 325-328 ofthe region consist of an amino acid motif selected from SAAF (SEQ ID NO:86), SKAF (SEQ ID NO: 87), NAAF (SEQ ID NO: 88) and NKAF (SEQ ID NO:89).

The altered antibodies of the invention are produced using any suitabletechnique including techniques that are well known to those skilled inthe art. For example, the altered antibodies are produced by modifyingknown antibodies to include at least one mutation in the Fc region,particularly in the CH2 domain, and more particularly at a locationselected from EU positions 325, 326 and 328. The numbering of theresidues in the gamma heavy chain is that of the EU index (see Edelman,G. M. et al., 1969; Kabat, E, A., T. T. Wu, H. M. Perry, K. S.Gottesman, and C. Foeller., 1991. Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed. U.S. Dept. of Health and Human Services, Bethesda,Md., NIH Publication n. 91-3242). The numbering for the immunoglobulinvariable regions for the antibodies described herein is as defined byE.A. Kabat et al., 1991. (Kabat, E, A., T. T. Wu, H. M. Perry, K. S.Gottesman, and C. Foeller., 1991. Sequences of Proteins of ImmunologicalInterest, 5^(th) Ed. U.S. Dept. of Health and Human Services, Bethesda,Md., NIH Publication n. 91-3242).

Pharmaceutical compositions according to the invention can include analtered antibody of the invention and a carrier. The altered antibodiescan equally be of murine, human and rat origin given the high sequencehomology between the different immunoglobulins. The composition caninclude a single isotype class, e.g., an IgG1 isotype altered antibody,or any combination of rat, mouse and human IgG isotype classes, e.g.,IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4 and combinations thereof.These pharmaceutical compositions can be included in kits, such as, forexample, diagnostic kits.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of mouse and chimeric IgG1 15C1antibodies. The mouse variable and constant domains of the light andheavy chain are in solid black. The human constant domains of light andheavy chains are in hatched black. Mouse IgG1 15C1 is a mouseimmunoglobulin of the IgG1 subclass specific for human TLR4. ChimericIgG1 15C1 is a recombinant immunoglobulin consisting of the mouse heavyand light chain variable regions of 15C1 in fusion with human IgG1 heavyand Kappa light constant regions. V=variable domain; L=light chain;H=heavy chain; CK=Kappa constant domain of the light chain; CH1, CH2,CH3=constant domains of the heavy chain.

FIG. 1B is a graph depicting LPS-dependent IL-8 production on humanembryonic kidney 293 cells (HEK 293) expressing human TLR4/MD2 by mouseand chimeric IgG1 15C1.

FIG. 2 is a graph depicting LPS-dependent IL-6 production in human wholeblood assay by mouse and chimeric IgG1 15C1 antibodies.

FIG. 3 is a graph depicting LPS-dependent IL-6 production in human wholeblood assay by recombinant mouse IgG1 15C1 and D265A mutant antibodies.

FIG. 4A Addition of either α-CD32 MAb AT10 or α-CD32A MAb IV.3diminished MAb 15C1 inhibition of LPS to a similar extent, as measuredby IL-6 production in whole blood.

FIG. 4B. 15C1 mediated blockade of LPS-dependent TLR4 activation inwhole blood derived from homozygous and heterozygous individuals usingmouse IgG1 or human IgG4 version of 15C1. Because the magnitude of IL-6production was variable among different donors, results are given as thepercentage of inhibition of IL-6 release compared to values obtainedwith the isotype control antibody (which corresponding to 100% LPSactivation). Errors bars show±SEM.

FIG. 5A is a schematic representation of chimeric and mouse IgG1 15C1antibodies containing the mouse and human IgG1 CH2 domains,respectively. The mouse variable and constant domains of the light andheavy chain are in black. The human constant domains of the light andheavy chains are in hatched black. Mouse IgG1 15C1 is a mouseimmunoglobulin of the IgG1 subclass specific for human TLR4. ChimericIgG1 15C1 is a recombinant immunoglobulin consisting of the mouse heavyand light chain variable regions of 15C1 in fusion with human IgG1 heavyand Kappa light constant regions. V=variable domain; L=light chain;H=heavy chain; CK=Kappa constant domain of the light chain; CH1, CH2,CH3=constant domains of the heavy chain.

FIG. 5B is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by swapping the CH2 domain between mouse and chimericIgG1 15C1 antibodies.

FIG. 6A is a schematic representation of four mouse CH2 mutants (A, B, Cand D) each containing the homologous corresponding human IgG1 CH2sub-region; residues 231-262, 318-340, 295-318 and 262-295 for mutantsA, B, C and D, respectively.

FIG. 6B is a graph depicting binding to CHO stable cell line expressinghuman TLR4-MD2 complex on their surface.

FIG. 6C is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by chimeric IgG1 15C1 containing hybrid CH2 sub-regiondomains between mouse and human IgG1.

FIG. 7A is a schematic representation of chimeric IgG1 15C1 containingthe mouse IgG1 CH2 domain residues 319 to 340. The mouse variable andconstant domains of the light and heavy chain are in black. The humanconstant domains of the light and heavy chains are in hatched black.Chimeric IgG1 15C1 is a recombinant immunoglobulin consisting of themouse heavy and light chain variable regions of 15C1 in fusion withhuman IgG1 heavy and Kappa light constant regions. V=variable domain;L=light chain; H=heavy chain; CK=Kappa constant domain of the lightchain; CH1, CH2, CH3=constant domains of the heavy chain.

FIG. 7B is a graph depicting LPS-dependent IL-6 production in a humanwhole blood assay by chimeric IgG1 15C1 or chimeric IgG1 15C1 containingeither the full length or mutant 319-340 mouse CH2 domain.

FIG. 8A is an illustration depicting an alignment of deduced amino-acidsequences of the mouse IgG1 C-terminal end (residue 319 to 340) of theheavy chain CH2 domain with human IgG1 C-terminal end (residue 319 to340) of the heavy chain CH2 domain. Dashes indicate amino acidsidentical with those in the mouse sequence. Sequences were aligned onthe basis of maximum nucleic acid alignment according to EU numbering.

FIG. 8B is an illustration depicting an alignment of deduced amino-acidsequences of the mouse IgG1 C-terminal end (residue 319 to 340) of theheavy chain CH2 domain with the 5 mutants (A to E) and human IgG1C-terminal end (residue 319 to 340) of the heavy chain CH2 domain.Dashes indicate amino acids identical with those in the mouse sequence.Sequences were aligned on the basis of maximum nucleic acid alignmentaccording to EU numbering. Mutations are denoted by the mouse amino acidresidue followed by a number from 1 to 7 corresponding to the sevendifferences between the mouse and human sequences and finally the humanamino acid it has been mutated to.

FIG. 8C is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by the chimeric IgG1 15C1, chimeric IgG1 15C1containing either the full length or mutants 319-340 mouse CH2.

FIG. 9A is a graph depicting binding to CHO stable cell line expressinghuman TLR4-MD2 on their surface.

FIG. 9B is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by chimeric IgG1 15C1, muCH2 15C1 and mutants chimericIgG1 15C1 antibodies.

FIG. 10 is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by the chimeric IgG1 15C1, mouse IgG1 15C1, chimericIgG1 15C1 containing mouse CH2 and humanized 15C1 containing mouse CH2.

FIG. 11A is a graph depicting Binding to CHO stable cell line expressinghuman TLR4-MD2 on their surface.

FIG. 11B is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by the humanized 15C1 mutants C, F, G and H andhumanized 15C1 containing the mouse CH2.

FIG. 12 is a graph depicting LPS-dependent IL-6 production in humanwhole blood assay by chimeric IgG1 15C1, humanized 15C1 mutant C andhumanized 15C1 containing mouse CH2.

FIGS. 13A-13C are a series of graphs depicting LPS-dependent IL-6production in human whole blood assay by mouse anti-TLR2 (13A), mouseanti-MD2 (18H10, 13B) and mouse anti-CD14 (13C) MAs with or withoutmouse anti-human CD32 monoclonal antibody.

FIG. 14A is an illustration depicting a nucleic acid sequence encodingthe accessory protein MD-2 (SEQ ID NO:43).

FIG. 14B is an illustration depicting an amino acid sequence of a matureMD-2 accessory protein (SEQ ID NO:44).

FIG. 15 is an illustration depicting the amino acid sequence of humantoll-like receptor 4 (TLR4) (SEQ ID NO:54).

FIG. 16 is an illustration depicting the protein display of the CH2domain of human, mouse and rat IgG isotypes. “*” means that the residuesin that column are identical in all sequences in the alignment.

FIGS. 17A-17G are a series of graphs depicting the analysis of 15C1humanized mutants by flow cytometry on cells expressing recombinanthuman TLR4-MD2.

DETAILED DESCRIPTION OF THE INVENTION

The altered antibodies described herein are antibodies that include atleast one specific amino acid substitution in the gamma heavy chainconstant region such that the altered antibody elicits alterations inantigen-dependent effector function while retaining binding to antigenas compared to an unaltered antibody. In a preferred embodiment, thealtered antibodies are human. For example, the altered antibodies areIgG1, IgG2, IgG3 or IgG4 isotype.

The altered antibodies of the invention also include an altered antibodyhaving a variant CDR3 region in which at least one amino acid residue inthe CDR3 region of the antibody has been modified. The alteredantibodies and altered polypeptide of the invention also includepolypeptides that include at least an FγR binding portion of an Fcregion of an immunoglobulin polypeptide and a variant CDR3 region. Thealtered antibodies and altered polypeptide of the invention also includepolypeptides that include at least a variant Fc region of animmunoglobulin polypeptide and a variant CDR3 region. The variant CDR3regions include the variant VH CDR3 regions shown in Example 4:KDPSDAFPY (SEQ ID NO: 80) and KDPSEGFPY (SEQ ID NO: 81). The variantCDR3 regions include the variant VL CDR3 regions shown in Example 4:QNSHSFPLT (SEQ ID NO: 82); QQGHSFPLT (SEQ ID NO: 83); QNSSSFPLT (SEQ IDNO: 84); and QQSHSFPLT (SEQ ID NO: 85).

The altered antibodies of the invention include an altered antibody inwhich at least the amino acid residue at EU position 328 in the CH2domain of the Fc portion of the antibody has been modified. For example,at least the amino acid residue at EU position 328 has been substitutedwith phenylalanine. In the altered antibodies described herein, at leastthe amino acid residue at EU position 328 alone or together with EUamino acid positions 325 and 326 are substituted with a differentresidue as compared to an unaltered antibody.

These altered antibodies with a modified Fc portion elicit modifiedeffector functions e.g., a modified Fc receptor activity, as compared toan unaltered antibody. For example, the human Fc receptor is CD32A. Insome embodiments, the altered antibodies elicit a prevention ofproinflammatory mediators release following ligation to CD32A ascompared to an unaltered antibody. Thus, the altered antibodiesdescribed herein elicit a modified Fc receptor activity, such as theprevention of proinflammatory mediators release while retaining theability to bind a target antigen. In some embodiments, the alteredantibody is a neutralizing antibody, wherein the altered antibodyelicits a modified Fc receptor activity, while retaining the ability toneutralize one or more biological activities of a target antigen.

For example, altered antibodies of the invention include monoclonalantibodies that bind the human TLR4/MD-2 receptor complex. This receptorcomplex is activated by lipopolysaccharide (LPS), the major component ofthe outer membrane of gram-negative bacteria. The altered antibodies ofthe invention inhibit receptor activation and subsequent intracellularsignaling via LPS. Thus, the altered antibodies neutralize theactivation of the TLR4/MD-2 receptor complex. In particular, theinvention provides altered antibodies that recognize the TLR4/1VD-2receptor complex expressed on the cell surface. These altered antibodiesblock LPS-induced IL-8 production. In addition, some altered antibodiesof the invention also recognize TLR4 when not complexed with MD-2. Thealtered antibody is, e.g., a humanized antibody.

Antibodies of the invention include antibodies that bind the humanTLR4/MD-2 receptor complex and also bind TLR4 independently of thepresence of MD-2. Antibodies of the invention also include antibodiesthat bind the TLR4 portion of the human TLR4/MD-2 receptor complex butbinding is dependent on the presence of MD-2, but binding is greatlyenhanced by the presence of MD-2, which suggests that the presence ofthe MD-2 causes a conformational change in TLR4, thereby exposing anepitope bound by the antibody. In addition, antibodies of the inventioninclude antibodies that bind the human TLR4/MD-2 receptor complex andalso bind MD-2 in the presence of TLR4.

Altered antibodies of the invention also include antibodies thatrecognize targets such as any toll-like receptor. Toll receptors, firstdiscovered in Drosophila, are type I transmembrane protein havingleucine-rich repeats (LRRs) in the extracellular portion of the protein,and one or two cysteine-rich domains. The mammalian homologs of theDrosophila Toll receptors are known as “Toll-like receptors” (TLRs).TLRs play a role in innate immunity by recognizing microbial particlesand activating immune cells against the source of these microbialparticles.

Currently, eleven types of Toll-like receptors have been identified inhumans, TLRs 1-11 (Pandey S and Agrawal D K, Immunobiology ofToll-like-receptors: emerging trends. Immunol. Cell Biol., 2006;84:333-341). These TLRs are characterized by the homology of theirintracellular domains to that of the IL-1 receptor, and by the presenceof extracellular leucine-rich repeats. The different types of TLRs areactivated by different types of microbial particles. For example, TLR4is primarily activated by lipopolysaccharide (LPS), while TLR2 isactivated by lipoteichoic (LTA), lipoarabinomannan (LAM); lipoprotein(BLP), and peptideglycans (PGN). Toll receptor homologs, such as RP105,have also been identified.

For example, altered antibodies of the invention include antibodies thatrecognize TLR2, including, e.g., one or more modified versions of theanti-TLR2 monoclonal antibody known as T2.5 (see e.g., WO 2005/028509,hereby incorporated by reference in its entirety).

Other suitable altered antibodies include antibodies that recognizeCD14, such as one or more modified versions of the anti-CD14 monoclonalantibody known as 28C5 (see e.g., U.S. Pat. No. 6,444,206, herebyincorporated by reference in its entirety).

Definitions:

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically bind” or“immunoreacts with” or “immunospecifically bind” is meant that theantibody reacts with one or more antigenic determinants of the desiredantigen and does not react with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶). Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and an F_(ab)expression library.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Ingeneral, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. For example, antibodies may be raisedagainst N-terminal or C-terminal peptides of a polypeptide. An antibodyis said to specifically bind an antigen when the dissociation constantis ≦1 μM; e.g., ≦100 nM, preferably ≦10 nM and more preferably ≦1 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smaller Karepresents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (Koff) can be determined by calculation of theconcentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to its target, when the equilibrium bindingconstant (K_(d)) is ≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, and morepreferably ≦1 nM, as measured by assays such as radioligand bindingassays or similar assays known to those skilled in the art.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Polynucleotides inaccordance with the invention include the nucleic acid moleculesencoding the heavy chain immunoglobulin molecules, and nucleic acidmolecules encoding the light chain immunoglobulin molecules describedherein.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Polypeptides in accordance with the invention comprise the heavychain immunoglobulin molecules, and the light chain immunoglobulinmolecules described herein, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as kappa light chainimmunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,6-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide- containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur- containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes r radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Antibodies

The altered antibodies described herein are antibodies that include atleast one specific amino acid substitution in the gamma heavy chainconstant region such that the altered antibody elicits alterations inantigen-dependent effector function while retaining binding to antigenas compared to an unaltered antibody. In a preferred embodiment, thealtered antibodies are human. For example, the altered antibodies areIgG1, IgG2, IgG3 or IgG4 isotype.

The altered antibodies of the invention also include an altered antibodyhaving a variant CDR3 region in which at least one amino acid residue inthe CDR3 region of the antibody has been modified. The alteredantibodies and altered polypeptide of the invention also includepolypeptides that include at least an FcγR binding portion of an Fcregion of an immunoglobulin polypeptide and a variant CDR3 region. Thealtered antibodies and altered polypeptide of the invention also includepolypeptides that include at least a variant Fc region of animmunoglobulin polypeptide and a variant CDR3 region. The variant CDR3regions include the variant VH CDR3 regions shown in Example 4:KDPSDAFPY (SEQ ID NO: 80) and KDPSEGFPY (SEQ ID NO: 81). The variantCDR3 regions include the variant VL CDR3 regions shown in Example 4:QNSHSFPLT (SEQ ID NO: 82); QQGHSFPLT (SEQ ID NO: 83); QNSSSFPLT (SEQ IDNO: 84); and QQSHSFPLT (SEQ ID NO: 85).

In one embodiment, altered antibodies that recognize TLR4, MD2 and/orthe TLR4/MD2 complex have the ability to inhibit LPS-inducedproinflammatory cytokine production. This inhibition is achieved via across-talk mechanism between the Fv portion of the altered antibodybinding to its target antigen while its modified Fc portion is engagingwith human CD32A. Inhibition is determined, for example, in the humanwhole blood and huTLR4/MD2 transfected HEK 293 cellular assays describedherein. In this embodiment, the altered antibody is, for example amodified version of the monoclonal antibodies referred to herein as“mu18H10”, “hu18H10”, “mu16G7”, “mu15C1”, “hu15C1”, “mu7E3” and “hu7E3”.The mu18H10 and hu18H10 antibodies recognize the TLR4/MD-2 complex, butdo not recognize an MD-2 protein when not complexed with TLR4. The mu16G7, mu15C1, hu15C1, mu7E3 and hu7E3 monoclonal antibodies recognizethe TLR4/MD-2 complex. mu15C1, hu15C1 and 16G7 also recognize TLR4 whennot complexed with MD-2.

Also included in the invention are antibodies that bind to the sameepitope as the altered antibodies described herein. For example, alteredantibodies of the invention specifically bind a TLR4/MD-2 complex,wherein the antibody binds to an epitope that includes one or more aminoacid residues on human TLR4 between residues 289 and 375 of the aminoacid sequence shown in FIG. 15. In another example altered antibodiesthat specifically bind the TLR4/MD2 complex, wherein the antibody bindsto an epitope on human MD-2 between residues 19 and 57 of the amino acidsequence shown in FIG. 14B. Those skilled in the art will recognize thatit is possible to determine, without undue experimentation, if amonoclonal antibody has the same specificity as an altered antibody ofthe invention by ascertaining whether the former prevents the latterfrom binding to the target (e.g., TLR2, CD14, TLR4/MD-2 complex or toTLR4 when not complexed to MD-2). If the monoclonal antibody beingtested competes with the altered antibody of the invention, as shown bya decrease in binding by the altered antibody of the invention, then thetwo antibodies bind to the same, or a closely related, epitope. Analternative method for determining whether a monoclonal antibody has thespecificity of an altered antibody of the invention is to pre-incubatethe altered antibody of the invention with the target with which it isnormally reactive, and then add the monoclonal antibody being tested todetermine if the monoclonal antibody being tested is inhibited in itsability to bind the target. If the monoclonal antibody being tested isinhibited then, in all likelihood, it has the same, or functionallyequivalent, epitopic specificity as the monoclonal antibody of theinvention.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a given target,such as, for example, a toll-like receptor, the TLR4/MD-2 complex, orTLR4 when not complexed to MD-2, TLR2, CD14, or against derivatives,fragments, analogs homologs or orthologs thereof. (See, for example,Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein byreference).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (April 17, 2000), pp. 25-28).

Preferably, the altered antibodies of the invention are monoclonalantibodies. Altered antibodies are generated, e.g., by immunizing BALB/cmice with combinations of cell transfectants expressing high levels of agiven target on their surface. Hybridomas resulting from myeloma/B cellfusions are then screened for reactivity to the selected target.

Monoclonal antibodies are prepared, for example, using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes can be immunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, California and the American Type CultureCollection, Manassas, Virginia. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (MA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Monoclonal antibodies of the invention include humanized antibodies orhuman antibodies. These antibodies are suitable for administration tohumans without engendering an immune response by the human against theadministered immunoglobulin. Humanized forms of antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a humanimmunoglobulin, and contain minimal sequence derived from a non-humanimmunoglobulin. Humanization is performed, e.g., by following the methodof Winter and co-workers (Jones et al., Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies also comprise, .e.g., residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody includes substantially allof at least one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin consensus sequence. The humanizedantibody optimally also includes at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)).

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanantibodies”, or “fully human antibodies” herein. Monoclonal antibodiescan be prepared by using trioma technique; the human B-cell hybridomatechnique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBVhybridoma technique to produce monoclonal antibodies (see Cole, et al.,1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc.,pp. 77-96). Monoclonal antibodies may be utilized and may be produced byusing human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA80: 2026-2030) or by transforming human B-cells with Epstein Barr Virusin vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. An example of such a nonhumananimal is a mouse termed the Xenomouse™ as disclosed in PCT publicationsWO 96/33735 and WO 96/34096. This animal produces B cells which secretefully human immunoglobulins. The antibodies can be obtained directlyfrom the animal after immunization with an immunogen of interest, as,for example, a preparation of a polyclonal antibody, or alternativelyfrom immortalized B cells derived from the animal, such as hybridomasproducing monoclonal antibodies. Additionally, the genes encoding theimmunoglobulins with human variable regions can be recovered andexpressed to obtain the antibodies directly, or can be further modifiedto obtain analogs of antibodies such as, for example, single chain Fv(scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds specifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA.gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for a target such as TLR4, MD2, TLR4/1VD2 complex,TLR2, CD14 or any toll-like receptor. The second binding target is anyother antigen, and advantageously is a cell-surface protein or receptoror receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface includes at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. The bispecificantibodies produced can be used as agents for the selectiveimmobilization of enzymes.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating diseases and disorders associated with aberrant LPSsignaling. For example, cysteine residue(s) can be introduced into theFc region, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated can have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). (See Caronet al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody can be engineered that hasdual Fc regions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies of theinvention. (See, for example, “Conjugate Vaccines”, Contributions toMicrobiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds),Carger Press, New York, (1989), the entire contents of which areincorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature. (See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat.No. 5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NETS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Altered Antibodies

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include an alteredantibody of the invention are used to treat or alleviate a symptomassociated with an immune-related disorder. The present invention alsoprovides methods of treating or alleviating a symptom associated with animmune-related disorder. A therapeutic regimen is carried out byidentifying a subject, e.g., a human patient suffering from (or at riskof developing) an immune-related disorder, using standard methods. Forexample, altered antibodies of the invention are useful therapeutictools in the treatment of autoimmune diseases and/or inflammatorydisorders. In certain embodiments, the use of altered antibodies thatmodulate, e.g., inhibit, neutralize, or interfere with, TLR signaling iscontemplated for treating autoimmune diseases and/or inflammatorydisorders.

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Ménière's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

For example, altered antibodies are useful in the treatment of acuteinflammation and sepsis induced by microbial products (e.g., LPS) andexacerbations arising from this acute inflammation, such as, forexample, chronic obstructive pulmonary disease and asthma (see O′Neill,Curr. Opin. Pharmacol. 3: 396-403 (2003), hereby incorporated byreference in its entirety). Such antibodies are also useful in treatingneurodegenerative autoimmune diseases. (Lehnardt et al., Proc. Natl.Acad. Sci. USA 100: 8514-8519(2003), hereby incorporated by reference inits entirety).

In addition, the antibodies of the invention are also useful astherapeutic reagents in the treatment of diseases, such as, for example,osteoarthritis, which are caused by mechanical stress, which, in turn,induces endogenous soluble “stress” factors that trigger TLR4.Endogenous soluble stress factor include e.g., Hsp60 (see Ohashi et al.,J. Immunol. 164: 558-561 (2000)) and fibronectin (see Okamura et al., J.Biol. Chem. 276: 10229-10233 (2001) and heparin sulphate, hyaluronan,gp96, β-Defensin-2 or surfactant protein A (see e.g., Johnson et al.,Crit. Rev. Immunol., 23(1-2):15-44 (2003), each of which is herebyincorporated by reference in its entirety). The antibodies of theinvention are also useful in the treatment of a variety of disordersassociated with mechanical stress, such as for example, mechanicalstress that is associated with subjects and patients placed onrespirators, ventilators and other respiratory-assist devices. Forexample, the antibodies of the invention are useful in the treatment ofventilator-induced lung injury (“VILI”), also referred to asventilation-associated lung injury (“VALI”).

Other disease areas in which inhibiting TLR4 function could bebeneficial include, for example, chronic inflammation (e.g., chronicinflammation associated with allergic conditions and asthma), autoimmunediseases (e.g., inflammatory bowel disorder) and atherosclerosis (seeO′Neill, Curr. Opin. Pharmacol. 3: 396-403 (2003), hereby incorporatedby reference in its entirety).

Symptoms associated with these immune-related disorders include, forexample, inflammation, fever, general malaise, fever, pain, oftenlocalized to the inflamed area, rapid pulse rate, joint pain or aches(arthralgia), rapid breathing or other abnormal breathing patterns,chills, confusion, disorientation, agitation, dizziness, cough, dyspnea,pulmonary infections, cardiac failure, respiratory failure, edema,weight gain, mucopurulent relapses, cachexia, wheezing, headache, andabdominal symptoms such as, for example, abdominal pain, diarrhea orconstipation.

Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular immune-relateddisorder. Alleviation of one or more symptoms of the immune-relateddisorder indicates that the antibody confers a clinical benefit.

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against a target such as TLR2, CD14, TLR4, MD2, theTLR4/MD-2 complex or any toll-like receptor (or a fragment thereof) maybe used in methods known within the art relating to the localizationand/or quantitation of these targets, e.g., for use in measuring levelsof these targets within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific any of these targets, orderivative, fragment, analog or homolog thereof, that contain theantibody derived antigen binding domain, are utilized aspharmacologically active compounds (referred to hereinafter as“Therapeutics”).

An altered antibody of the invention can be used to isolate a particulartarget using standard techniques, such as immunoaffinity, chromatographyor immunoprecipitation. Altered antibodies of the invention (or afragment thereof) can be used diagnostically to monitor protein levelsin tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may be used as therapeutic agents. Suchagents will generally be employed to treat or prevent a disease orpathology associated with aberrant expression or activation of a giventarget in a subject. An antibody preparation, preferably one having highspecificity and high affinity for its target antigen, is administered tothe subject and will generally have an effect due to its binding withthe target. Administration of the antibody may abrogate or inhibit orinterfere with the signaling function of the target. Administration ofthe antibody may abrogate or inhibit or interfere with the binding ofthe target with an endogenous ligand to which it naturally binds. Forexample, the antibody binds to the target and neutralizes LPS-inducedproinflammatory cytokine production.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies or a fragment thereof of the invention can be administeredfor the treatment of a variety of diseases and disorders in the form ofpharmaceutical compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) MackPub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,Possibilities, Limitations, And Trends, Harwood Academic Publishers,Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of a given target (or a protein fragment thereof)in a sample. In some embodiments, the antibody contains a detectablelabel. Antibodies are polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., F_(ab), scFv, orF_((ab)2)) is used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or soluble chimeric polypeptides of the invention (alsoreferred to herein as “active compounds”), and derivatives, fragments,analogs and homologs thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the antibody or soluble chimeric polypeptide and apharmaceutically acceptable carrier. As used herein, the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Generation of Monoclonal Antibodies

The following studies describe the use of antibodies that recognize anepitope on TLR4, MD2 and/or the TLR4/MD2 complex. The antibodies used inthe studies presented herein were generated using the methods describedin co-pending U.S. application Ser. Nos. 11/009,939, filed Dec. 10, 2004and Ser. No. 11/151916, filed Jun. 15, 2004 and in WO 05/065015, filedDec. 10, 2004 and PCT/US2005/020930, filed Jun. 15, 2004, each of whichis hereby incorporated by reference in its entirety.

The amino acid and nucleic acid sequences of the heavy chain variable(VH) and light chain variable (VL) regions of the anti-TLR4/MD2antibodies are shown below. The amino acids encompassing thecomplementarity determining regions (CDR) as defined by Chothia et al.1989, E. A. Kabat et al., 1991 are highlighted in underlined anditalicized text below. (See Chothia, C, et al., Nature 342:877-883(1989); Kabat, EA, et al., Sequences of Protein of immunologicalinterest, Fifth Edition, US Department of Health and Human Services, USGovernment Printing Office (1991)).

1 caggtgcaac tgcagcagtc tggggctgat cttgtgaggc caggggcctt    q  v  q   l  q  q  s   g  a  d   l  v  r   p  g  a 51agtcaagttg tcctgcacag cttctggctt caacattaaa gactcctatal  v  k  l   s  c  t   a  s  g  f   n  i  k   d  s  y 101tacactgggt gaagaagagg cctgaatggg gcctggagtg gattggatgg i  h  w  v   k  k  r   p  e  w   g  l  e  w   i  g  w 151actgatcctg agaatgttaa ttctatatat gacccgaggt ttcagggcaa  t  d  p   e  n  v  n   s  i  y   d  p  r   f  q  g 201ggccagtata acagcagaca catcctccaa cacagccttc cttcagctcak  a  s  i   t  a  d   t  s  s   n  t  a  f   l  q  l 251ccagcctgac atctgaggac actgccgtct attactgtgc taggggttat t  s  l   t  s  e  d   t  a  v   y  y  c   a  r  g  y 301aacggtgttt actatgctat ggactactgg ggccaaggga cctcagtcac  n  g  v   y  y  a   m  d  y  w   g  q  g   t  s  v 351cgtctcctca (SEQ ID NO: 1) t  v  s  s (SEQ ID NO: 2)

18H10 VH Protein sequence

(SEQ ID NO: 2) 1 qvqlqqsgad lvrpgalvkl sctasgfnik 

wvkkr pewglewig

51

 

kasi tadtssntaf lqltsltsed tavyycar

101

w gqgttvtvss

18H10 VH CDR Protein Sequences

(SEQ ID NO: 3) dsyih (SEQ ID NO: 4) wtdpenvnsiydprfgg (SEQ ID NO: 5)gyngvyyamdy

18H10 VL Nucleotide Sequence

1 caaattgttc tcacccagtc tccatcaatc atgtctgcgt ctctagggga    q  i  v   l  t  q   s  p  s  i   m  s  a   s  l  g 51ggagatcacc ctaacctgca gtgccagctc gagtgtaatt tacatgcacte  e  i  t   l  t  c   s  a  s   s  s  v  i   y  m  h 101ggtaccagca gaagtcaggc acttctccca aactcttgat ttataggaca w  y  q   q  k  s  g   t  s  p   k  l  l   i  y  r  t 151tacaacctgg cttctggagt cccttctcgc ttcagtggca gtgggtctggy  n  l   a  s  g   v  p  s  r   f  s  g   s  g  s 201gaccttttat tctctcacaa tcagcagtgt ggaggctgaa gatgctgccgg  t  f  y   s  l  t   i  s  s   v  e  a  e   d  a  a 251attattactg ccatcagtgg agtagttttc cgtacacgtt cggagggggg d  y  y   c  h  q  w   s  s  f   p  y  t   f  g  g  g 301accaagctgg aaatcaaacg g (SEQ ID NO: 6)  t  k  l   e  i  k  r (SEQ ID NO: 7)

18H10 VL Protein Sequence

(SEQ ID NO: 7) 1 qviltqspsi msaslgeeit ltc

 

wyqqksg tspklliy

51

gvpsr fsgsgsgtfy sltissveae daadyyc

 

fggg 101 tkleikr

18H10 VL CDR Protein Sequences

(SEQ ID NO: 8) sasssviymh (SEQ ID NO: 9) rtynlas (SEQ ID NO: 10)hqwssfpyt

16G7 VH Nucleotide Sequence

1 aggtgaaact gcaggagtct ggagctgagc tgatgaagcc tggggcctca    v  k  l   q  e  s   g  a  e   l  m  k   p  g  a  s 51gtgaagatat cctgcaaggc tactggctac aaattcagtg actactggat  v  k  i   s  c  k   a  t  g  y   k  f  s   d  y  w 101agagtggata aaacagaggc ctggacatgg ccttgagtgg attggagagai  e  w  i   k  q  r   p  g  h   g  l  e  w   i  g  e 151ttttgcctgg aagtggtagt actaactaca atgaggactt caaggacaag i  l  p   g  s  g  s   t  n  y   n  e  d   f  k  d  k 201gccacattca cttcagatac atcctccaac acagcctaca tgcaactcag  a  t  f   t  s  d   t  s  s  n   t  a  y   m  q  l 251cagcctgaca tctgaagact ctgccgtcta ttactgtgca aaagaggagas  s  l  t   s  e  d   s  a  v   y  y  c  a   k  e  e 301gggcgtacta ctttggctat tggggccaag ggaccacggt caccgtctcc r  a  y   y  f  g  y   w  g  q   g  t  t   v  t  v  s 351tca (SEQ ID NO: 11)   s (SEQ ID NO: 12)

16G7 VH Protein Sequence

(SEQ ID NO: 12) 1 qvqlqqsgaelmkpgasvkisckatgykfs

wikqrpghglewig

51

katftsdtssntaymqls sltsedsavyycak

101

wgqgttvtvss

16G7 VH CDR Protein Sequences

(SEQ ID NO: 13) dywie (SEQ ID NO: 14) eilpgsgstnynedfkd (SEQ ID NO: 15)eerayyfgy

16G7 VL Nucleotide Sequence

1 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga  d  v  l   m  t  q   t  p  l  s   l  p  v   s  l  g 51tcaagcctcc atctcttgca ggtctagtca gagccttgaa aacagtaatgd  q  a  s   i  s  c   r  s  s   q  s  l  e   n  s  n 101gaaacaccta tttgaactgg tacctccaga aaccaggcca gtctccacag g  n  t   y  l  n  w   y  l  q   k  p  g   q  s  p  q 151ctcctgatct acagggtttc caaccgattt tctggggtcc tagacaggtt  l  l  i   y  r  v   s  n  r  f   s  g  v   l  d  r 201cagtggtagt ggatcaggga cagatttcac actgaaaatc agcagagtggf  s  g  s   g  s  g   t  d  f   t  l  k  i   s  r  v 251aggctgagga tttgggagtt tatttctgcc tccaagttac acatgtccct e  a  e   d  l  g  v   y  f  c   l  q  v   t  h  v  p 301cccacgttcg gtgctgggac caagctggaa ctgaaacgg (SEQ ID NO: 16)  p  t  f   g  a  g   t  k  l  e   l  k  r (SEQ ID NO: 17)

16G7 VL Protein Sequence

(SEQ ID NO: 17) 1 dvvmtqtplslpvslgdqasisc

wyl gkpgqspq 51 lliy

gvldrfsgsgsgtdftlkisrveaedlgvyfc

101

fgagtklelkr

16G7 VL CDR Protein Sequences

(SEQ ID NO: 18) rssqslensngntyln (SEQ ID NO: 19) rvsnrfs (SEQ ID NO: 20)lqvthvppt

15C1 VH Nucleotide Sequence

1gatgtgcagc ttcaggagtc aggacctgac ctaatacaac cttctcagtc actttcactc acctgcactg  d  v  q   l  q  e   s  g  p  d   l  i  q   p  s  q   s  l  s  l   t  c  t71tcactggcta ctccatcacc ggtggttata gctggcactg gatccggcag tttccaggaa acaaactgga v  t  g   y  s  i  t   g  g  y   s  w  h   w  i  r  q   f  p  g   n  k  l141atggatgggc tacatccact acagtggtta cactgacttc aacccctctc tcaaaactcg aatctctatce  w  m  g   y  i  h   y  s  g   y  t  d  f   n  p  s   l  k  t   r  i  s  i211actcgagaca catccaagaa ccagttcttc ctgcagttga attctgtgac tactgaagac acagccacat  t  r  d   t  s  k   n  q  f  f   l  q  l   n  s  v   t  t  e  d   t  a  t281attactgtgc aagaaaagat ccgtccgacg gatttcctta ctggggccaa gggactctgg tcactgtctc y  y  c   a  r  k  d   p  s  d   g  f  p   y  w  g  q   g  t  l   v  t  v351 tgca (SEQ ID NO: 21) s  a (SEQ ID NO: 22)

15C1 VH Protein Sequence

(SEQ ID NO: 22) 1 dvqlqesgpd liqpsqslsl tctvtgysit 

 wirq  fpgnklewmg 51

 

 

 risi trdtsknqff lqlnsvt  ted tatyycar 

101

 wgq gtlvtvsa

15C1 VH CDR Protein Sequences

(SEQ ID NO: 23) ggyswh (SEQ ID NO: 24) yihysgytdfnpslkt (SEQ ID NO: 25)kdpsdgfpy

15C1 VL Nucleotide Sequence

1 gacattgtga tgacccagtc tccagccacc ctgtctgtga ctccaggtga tagagtctct  d  i  v   m  t  q   s  p  a  t   l  s  v   t  p  g   d  r  v  s 61ctttcctgca gggccagcca gagtatcagc gaccacttac actggtatca acaaaaatca  l  s  c   r  a  s   q  s  i  s   d  h  l   h  w  y   q  q  k  s 121catgagtctc cacggcttct catcaaatat gcttcccatg ccatttctgg gatcccctcc  h  e  s   p  r  l   l  i  k  y   a  s  h   a  i  s   g  i  p  s 181aggttcagtg gcagtggatc agggacagat ttcactctca gcatcaaaag tgtggaacct  r  f  s   g  s  g   s  g  t  d   f  t  l   s  i  k   s  v  e  p 241gaagatattg gggtgtatta ctgtcaaaat ggtcacagtt ttccgctcac gttcggtgct  e  d  i   g  v  y   y  c  q  n   g  h  s   f  p  l   t  f  g  a 301gggaccaagc tggagctgaa a (SEQ ID NO: 26)  g  t  k   l  e  l   k (SEQ ID NO: 27)

15C1 VL Protein Sequence

(SEQ ID NO: 27) 1 divmtqspat lsvtpgdrvs lsc 

 

 wyqqks  hesprllik 

51

 gips rfsgsgsgtd ftlsiksvep edigvyyc 

 

 fga 101 gtklelkr

15C1 VL CDR Protein Sequences

(SEQ ID NO: 28) rasqsisdhlh (SEQ ID NO: 29) yashais (SEQ ID NO: 30)qnghsfplt

7E3 VH Nucleotide Sequence

1caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg acttgttctt  q  v  t   l  k  e   s  g  p  g   i  l  q   p  s  q   t  l  s  l   t  c  s71tctctgggtt ttcactgacc acttataata taggagtagg ctggattcgt cagccttcag ggaagggtct f  s  g   f  s  l  t   t  y  n   i  g  v   g  w  i  r   q  p  s   g  k  g141ggagtggctg gcacacattt ggtggaatga taatatttac tataatacag tccttaagag ccgactcacal  e  w  l   a  h  i   w  w  n   d  n  i  y   y  n  t   v  l  k   s  r  l  t211ttctccaagg atacctccaa caaccaggtt ttcctcaaga tcgccagtgt ggacattgca gatactgcca  f  s  k   d  t  s   n  n  q  v   f  l  k   i  a  s   v  d  i  a   d  t  a281catattactg tattcgaatg gctgagggaa ggtacgacgc tatggactac tggggtcaag gaacctcagt t  y  y   c  i  r  m   a  e  g   r  y  d   a  m  d  y   w  g  q   g  t  s351 caccgtctcc tca (SEQ ID NO: 31) v  t  v  s   s (SEQ ID NO: 32)

7E3 VH Protein Sequence

(SEQ ID NO: 32) 1 qvtlkesgpg ilqpsqtlsl tcsfsgfslt 

 wir qps gkglewl 51 a 

 

 

 rlt fskdtsnnqv flkiasvdia  dtatyycir 

101

 

 wgqgtsvtvs s

7E3 VH CDR protein Sequences

(SEQ ID NO: 33) tynigvg (SEQ ID NO: 34) hiwwndniyyntvlks (SEQ ID NO: 35)maegrydamdy

7E3 VL Nucleotide Sequence

1gctatccaga tgacacagag tacatcctcc ctgtctgcct ctctgggaga cagagtcacc atcaattgca  a  i  q   m  t  q   s  t  s  s   l  s  a   s  l  g   d  r  v  t   i  n  c71gggcaagtca ggacatcacc aattatttaa attggtatca gcagaaacca gatggaactg tcagactcct r  a  s   q  d  i  t   n  y  l   n  w  y   q  q  k  p   d  g  t   v  r  l141gatctattat acatcaaaat tacactcagg agccccatca aggttcagtg gccgtgggtc tggaacagatl  i  y  y   t  s  k   l  h  s   g  a  p  s   r  f  s   g  r  g   s  g  t  d211tattctctca ccattagtaa cctggagcaa gaggatattg ccacttactt ttgccaacag ggtaatacgt  y  s  l   t  i  s   n  l  e  q   e  d  i   a  t  y   f  c  q  q   g  n  t281 ttccgtggac gttcggtgga ggcaccaaac tggaaatcaa acgt (SEQ ID NO: 36) f  p  w   t  f  g  g   g  t  k   l  e  i   k  r (SEQ ID NO: 37)

7E3 VL Protein Sequence

(SEQ ID NO: 37) 1 aiqmtqstss lsaslgdrvt inc 

 

  wyqqkp dgtvrlliy 

51

 gaps rfsgrgsgtd ysltisnleq ediatyfc

 

 fgg 101 gtkleikr

7E3 VL CDR Protein Sequences

(SEQ ID NO: 38) rasqditnyln (SEQ ID NO: 39) ytsklhs (SEQ ID NO: 40)qqgntfpwt

The ability of each monoclonal antibody to neutralize LPS-induced IL-8induction on TLR4/MD2 transfected cells was analyzed by pre-incubatingthe transfected cells with each monoclonal antibody for 30 minutes priorto LPS administration. In addition, each monoclonal antibody was testedfor the ability to neutralize LPS-induced IL-8 induction in whole blood.

The specificity of each monoclonal antibody was tested by evaluating thebinding of each monoclonal antibody to cells transfected with thefollowing combinations: (1) human TLR4 and human MD-2; (2) rabbit TLR4and rabbit MD-2; (3) human TLR4 and rabbit MD-2; (4) rabbit TLR4 andhuman MD-2.

Example 2 Humanization of Murine Monoclonal Antibodies

The following studies describe the humanization of antibodies thatrecognize an epitope on TLR4, MD2 and/or the TLR4/MD2 complex. Theantibodies were humanized using the methods described in co-pending U.S.application Ser. No. 11/151916, filed Jun. 15, 2004 (U.S. PatentPublication No. US 2008-0050366 A1) and in PCT/IB2005/004206, filed Jun.15, 2004 (PCT Publication No. WO 07/110,678), each of which is herebyincorporated by reference in its entirety.

The hu15C1 antibodies include the variable heavy chain (V_(H)) 4-28shown below in SEQ ID NO:45 or the V_(H) 3-66 shown below in SEQ IDNO:46. The hu15C1 antibodies include the variable light chain (V_(L)) L6shown below in SEQ ID NO:47 or A26 shown below in SEQ ID NO:48. Theamino acids encompassing the complementarity determining regions (CDR)as defined by Chothia et al. 1989, E.A. Kabat et al., 1991 are boxed inthe sequences provided below. (See Chothia, C, et al., Nature342:877-883 (1989); Kabat, E A, et al., Sequences of Protein ofimmunological interest, Fifth Edition, US Department of Health and HumanServices, US Government Printing Office (1991)).

15C1 Hu V_(H) Version 4-28

(SEQ ID NO: 45)

(SEQ ID NO: 23) CDR 1: GGYSWH (SEQ ID NO: 24) CDR 2: YIHYSGYTDFNPSLKT(SEQ ID NO: 25) CDR 3: KDPSDGFPY Where X₁ is Thr or SerWhere X₂ is Ile or Met Where X₃ is Val or Ile Where X₄ is Met or Ile

15C1 Hu V_(H) Version 3-66

(SEQ ID NO: 46)

(SEQ ID NO: 23) CDR 1: GGYSWH (SEQ ID NO: 24) CDR 2: YIHYSGYTDFNPSLKT(SEQ ID NO: 25) CDR 3: KDPSDGFPY Where X₁ is Ala or ValWhere X₂ is Val or Met Where X₃ is Leu or Phe

15C1 Hu VL Version L6

(SEQ ID NO: 47)

(SEQ ID NO: 28) CDR1: RASQSISDHLH (SEQ ID NO: 29) CDR2: YASHAIS(SEQ ID NO: 30) CDR3: QNGHSFPLT Where X₁ is Lys or Tyr

15C1 Hu VL Version A26

(SEQ ID NO: 48)

(SEQ ID NO: 28) CDR1: RASQSISDHLH (SEQ ID NO: 29) CDR2: YASHAIS(SEQ ID NO: 30) CDR3: QNGHSFPLT

The hu18H10 antibodies include the V_(H) 1-69 shown below in SEQ IDNO:49. The hu18H10 antibodies include the V_(L) L6 shown below in SEQ IDNO:50. The amino acids encompassing the complementarity determiningregions (CDR) as defined by Chothia et al. 1989, E. A. Kabat et al.,1991 are boxed in the sequences provided below. (See Chothia, C, et al.,Nature 342:877-883 (1989); Kabat, E A, et al., Sequences of Protein ofimmunological interest, Fifth Edition, US Department of Health and HumanServices, US Government Printing Office

18H10 Hu VH Version 1-69

(SEQ ID NO: 49)

(SEQ ID NO: 3) CDR1: DSYIH (SEQ ID NO: 4 SEQ ID NO: 4)CDR2: WTDPENVNSIYDPRFQG (SEQ ID NO: 5) CDR3: GYNGVYYAMDYWhere X₁ is Met or Ile Where X₂ is Lys or Thr Where X₃ is Met or Leu

18H10 Hu VL Version L6

(SEQ ID NO: 50)

(SEQ ID NO: 8) CDR1: SASSSVIYMH (SEQ ID NO: 9) CDR2: RTYNLAS(SEQ ID NO: 10) CDR3: HQWSSFPYT Where X₁ is Phe or Tyr

The hu7E3 antibodies include the V_(H) 2-70 shown below in SEQ ID NO:51or the V_(H) 3-66 shown below in SEQ ID NO:52. The hu7E3 antibodiesinclude the V_(L) L19 shown below in SEQ ID NO:53. The amino acidsencompassing the complementarity determining regions (CDR) as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are boxed in the sequencesprovided below. (See Chothia, C, et al., Nature 342:877-883 (1989);Kabat, E A, et al., Sequences of Protein of immunological interest,Fifth Edition, US Department of Health and Human Services, US GovernmentPrinting Office (1991)).

7E3 Hu VH Version 2-70

(SEQ ID NO: 51)

(SEQ ID NO: 33) CDR1: TYNIGVG (SEQ ID NO: 34) CDR2: HIWWNDNIYYNTVLKS(SEQ ID NO: 35) CDR3: MAEGRYDAMDY Where X₁ is Ser or ThrWhere X₂ is Ile or Phe Where X₃ is Ile or Ala

7E3 Hu VH Version 3-66

(SEQ ID NO: 52)

(SEQ ID NO: 33) CDR1: TYNIGVG (SEQ ID NO: 34) CDR2: HIWWNDNIYYNTVLKS(SEQ ID NO: 35) CDR3: MAEGRYDAMDY Where X₁ is Phe or AlaWhere X₂ is Val or Leu Where X₃ is Ile or Phe Where X₄ is Lys or ArgWhere X₅ is Leu or Val Where X₆ is Ile or Ala

7E3 Hu VL version L19

(SEQ ID NO: 53)

(SEQ ID NO: 38) CDR1: RASQDITNYLN (SEQ ID NO: 39) CDR2: YTSKLHS(SEQ ID NO: 40) CDR3: QQGNTFPWT Where X₁ is Phe or TyrWhere X₂ is Tyr or Phe

The chimeric antibodies described above in Example 1 were used toevaluate the ability of the humanized monoclonal antibodies to bind tothe human TLR4/1VD2 complex. Each of the humanized monoclonal antibodieswas found to bind TLR4/MD2 in a similar manner to the correspondingchimeric antibody. In addition, the chimeric antibodies were used toevaluate the ability of the humanized monoclonal antibodies to inhibitLPS-induced IL-6 production in human whole blood. Each of the humanizedmonoclonal antibodies was found to inhibit the effects of LPS on bloodleukocytes in a similar manner to the corresponding chimeric antibody.

Example 3 Increasing the Potency of Modified Monoclonal Antibodies

The studies described herein are directed methods of increasing thepotency of neutralizing antibodies by modifying one or more residues inthe Fc portion of an antibody. In particular, the studies describedherein use an altered neutralizing antibody that recognizes the TLR4/MD2complex. These anti-TLR4/MD2 antibodies are modified to include one ormore mutations in the Fc portion, specifically in the CH2 domain of theFc portion.

The murine IgG1/K anti-human TLR4/MD2 monoclonal antibody discussedabove and referred to herein as “mu15C1” was modified by replacing themouse constant regions of mu15C1 with those of a human IgG1 to produce achimeric antibody, referred to herein as “chimeric IgG1 15C1” (FIG. 1A).The relative binding affinity of the corresponding MAbs was unchanged.

The ability of the chimeric IgG1 15C1 antibody to neutralize the effectsof LPS was evaluated using the TLR4/MD-2 transfected cell line. HEK 293cells were plated in 96-well plates at 6×10⁴ cells/well. The medium wasremoved on the day of the experiment and 30 μl of medium containing 6%heat-inactivated human plasma was added. Mouse IgG1 15C1 (square) orchimeric IgG1 15C1 (triangle) MAbs were diluted in 30 μl basal medium tothe appropriate concentration, and added to the cells for 1 hour at 37°C. LPS was diluted in 30 μl medium, added to the cells and left toincubate for 24 hours at 37° C. IL-8 secretion in the culturesupernatant was monitored by ELISA (Endogen).

The chimeric antibody was able to neutralize the effects of LPS on theTLR4/MD-2 transfected cell line HEK 293 (as measured by IL-8production). FIG. 1B shows that 15C1 on a mouse IgG1 backbone (referredas mouse IgG1 15C1; see schematic description in FIG. 1A) and 15C1 on ahuman IgG1 backbone (referred as chimeric IgG1 15C1; see schematicdescription in FIG. 1A) are equivalent in their neutralizing capacity onthis cell line.

The ability of the chimeric antibody to neutralize the effects of LPS inhuman whole blood was also evaluated. Fresh heparinated blood fromhealthy volunteers was obtained by venipuncture and diluted 1:2 withRPMI 1640. The diluted blood was plated at 60 μl/well in a 96-well plateand incubated for 15 minutes at 37° C. Then 30 μl of serial dilutions inRPMI 1640 of the mouse and chimeric IgG1 15C1 MAbs were added to theblood and incubated for an hour at 37° C. Blood cells were thenstimulated by adding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI1640 containing 0.1% HSA) to the wells and incubated for 6 hours. IL-6production was then measured by ELISA.

In the human whole blood assay, a different profile for the murine andchimeric antibodies was seen. FIG. 2 shows that mouse IgG1 15C1 wassignificantly more potent in its ability to neutralize LPS than chimericIgG1 15C1 (as measured by IL-6 production). This observation was morestriking the lower the concentration of MAb used in the assay. It wasconcluded that this difference had to be attributed to the Fc region ofthe molecule, as the binding affinity of the two MAbs (typicallyinvolving the Fab region) was equivalent.

Furthermore, it was thought that this difference was mediated via an Fcreceptor-dependent mechanism. On HEK 293 cells (FIG. 1B) negative for Fcgamma receptors expression, the two MAbs, chimeric and murine 15C1, wereequally potent whereas in an ex-vivo human whole blood assay, whereleucocytes are positive for Fc gamma receptors expression, a cleardifference was seen between them.

To further demonstrate the involvement of interactions between the MAbFc portion and Fcγ receptors in a putative inhibitory response, amodification of mouse IgG1 15C1 was engineered to disrupt the ability ofthe antibody to engage cellular Fcγ receptors while retaining itsaffinity for its cognate antigen TLR4/MD2. The mutation of Asp to Ala atEU amino acid residue position 265 (D265A) was introduced into the mouseIgG1 15C1 gamma heavy chain gene, and this mutated sequence wasexpressed along with the 15C1 mouse kappa chain in PEAK cells to producemutant (D265A) antibodies. In parallel recombinant wild type 15C1 heavyand light chains were co-expressed in PEAK cells to produce recombinantmouse IgG1 15C1. The mutation D265A when engineered in a mouse IgG1isotype (IgG1-D265A) has been shown to anneal the binding ofIgG1-D265A-containing immune complexes to mouse Fc-gamma receptors IIB(FcγRIIB), III (FcγRIII) and IV (FγRIV) (Nimmerjahn et al., Immunity,2005, 23:41-51) as well as to nullify (.FcγRI) or greatly reduce itsbinding to all human Fc gamma receptors (FcγRIIA, FcγRIIB and FcγRIII).(Shields et al., JBC, 2001; 276:6591-6604).

The neutralizing capability of the recombinant mouse IgG1 15C1 andrecombinant mouse IgG1-D265A 15C1 antibodies were evaluated in the humanwhole blood assay. In particular, fresh heparinated blood from healthyvolunteers was obtained by venipuncture and diluted 1:2 with RPMI 1640.The diluted blood was plated at 60 μl/well in a 96-well plate andincubated for 15 minutes at 37° C. Then 30 μl of serial dilutions inRPMI 1640 of recombinant mouse IgG1 15C1 (rec-mouse IgG1 15C1) andrecombinant mouse IgG1 15C1 containing the mutation Asp to Ala at EUposition 265 (rec-mouse D265A 15C1) MAbs were added to the blood andincubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

In a human whole blood experiment (FIG. 3) recombinant mouse D265A IgG1(rec-D265A mouse IgG1 15C1) was significantly less potent at inhibitingthe effects of LPS (as measured by IL-6 production) than recombinantmouse IgG1 15C1 WT IgG1 (rec mouse IgG1 15C1). This result confirmed thehypothesis that binding of the Fc portion of mouse IgG1 to human Fcγreceptors contributes to the potency of 15C1 to neutralize LPSpro-inflammatory stimulation.

Mouse IgG1 has been described in the literature to have a high affinityfor the human FcγRII or CD32. Indeed, this high affinity of mouse IgG1for human CD32 rendered the discovery of this receptor possible. It was,therefore, hypothesized that CD32 would be the key target receptor forthe Fc of mouse IgG1 on human leukocytes. In order to verify thispossibility, a human whole blood assay was performed using two differentanti-human CD32 antibody (AT10 which recognizes equally well CD32A andCD32B; and IV.3 which binds to CD32A and weakly to CD32B) in order toprevent the binding of the Fc of mouse IgG1 to this receptor. Freshheparinated blood from healthy volunteers was obtained by venipunctureand diluted 1:2 with RPMI 1640. The diluted blood was plated at 60μl/well in a 96-well plate and incubated for 15 minutes at 37° C. Then30 μl of serial dilutions in RPMI 1640 of the mouse IgG1 15C1 MAbs withor without mouse anti-human CD32 monoclonal antibody (Clone AT10, mouseIgG1, Catalog number MCA1075XZ, AbD Serotec, clone IV.3, mouse IgG2b,Catalog number 01470, StemCell Technologies) were added to the blood andincubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

In a human whole blood assay it was demonstrated (FIG. 4A) that bothAT10 and IV.3 anti-human CD32 antibodies reduce the potency of mouseIgG1 15C1 to a similar level to that seen with chimeric IgG1 15C1. Thefact that IV.3 binds strongly to CD32A, recognizes both phenotypic formsof FcγRIIA equally well and weakly to CD32B, point to the involvement ofhuman CD32A rather than CD32B.

CD32A contains a polymorphism (histidine or arginine) in itsextracellular domain at amino acid 131. The nature of this polymorphismhas an influence on the binding of mouse IgG1 to CD32A, with argininehomozygous individuals having a much higher affinity for mouse IgG1 thanhistidine homozygotes. Arginine/histidine heterozygotes have anintermediate affinity (Dijstelbloem, H. M. et al., 2001. Trends Immunol.22:510-516). We screened healthy individuals for their CD32A genotype atthis polymorphism and tested 15C1-mediated blockade of LPS-dependentTLR4 activation in whole blood derived from homozygous and heterozygousindividuals. For this experiment, 15C1 was produced in PEAK cells eitherin its original form (i.e. on a mouse IgG1 backbone) or as a chimericMAb with the 15C1 variable region on a human IgG4 backbone. This formatwas chosen as human IgG4 is known to have a very poor affinity for CD32.Following protein A affinity chromatography purification, the integrityof both MAbs for TLR4/MD-2 binding on transfected CHO cells wasconfirmed and shown to be equivalent (data not shown). In whole bloodLPS-activation experiments, the mIgG1 version of 15C1 was considerablymore potent at inhibiting TLR4 than the hIgG4 version when testingArg/Arg and Arg/His donors. In contrast, mIgG1 15C1 was only slightlymore potent that hIgG4 15C1 in His/His donors (FIG. 4B). IC50 valuesobtained for each dose response curve are shown in table 3. These valueshighlight the differences in potency between the 15C1mIgG1 and hIgG4constructs for the different CD32 genotype donors. The mIgG1 constructshows a dramatic loss in potency from the Arg/Arg to the His/His donors(˜35-fold), whereas the hIgG4 construct shows a marginal loss in potency(˜3 fold). These results reinforce the contribution of CD32A to theinhibitory activities of the 15C1 MAb on TLR4 signaling.

Identical results were obtained in human whole blood from the samedonors using heat-inactivated E. coli as a stimulus (10⁶ cfu/ml, datanot shown).

Studies were then designed to determine the critical region within theFc region of a mouse IgG1 antibody that is sufficient to maintain thebiological contribution of the whole Fc region. It is known in theliterature that both CH2 and CH3 domains of the Fc region can contributedirectly to the interaction with Fcγ receptors. In a first step towardsthe identification of the critical region contributing to thisinhibitory effect, studies were designed to focus on the importance ofthe CH2 domain (EU positions 231 to 340, see alignment of CH2 domains inFIG. 16) since its role in the interaction of the Fc with Fcγ receptorsis well documented. The CH2 domain of human IgG1 was replaced with thatof mouse IgG1 (later referred as muCH2-chimeric IgG1 15C1; see FIG. 5A)and reciprocally the CH2 domain of mouse IgG1 was replaced with that ofhuman IgG1 (later referred as huCH2-mouse IgG1 15C1; see FIG. 5A). Theantibodies were produced in PEAK cells and purified fromtransfected-cell supernatants by protein G affinity columnchromatography. Equivalent binding to CHO TLR4/MD2 transfectants by FACSwas observed indicating that the change of CH2 domain had no significantinfluence on the relative affinity for the antigen. The neutralizingcapability of murine 15C1, huCH2-mouse IgG1 15C1, chimeric IgG1 15C1 andmuCH2-chimeric IgG1 15C1 was evaluated in the human whole blood assay.Fresh heparinated blood from healthy volunteers was obtained byvenipuncture and diluted 1:2 with RPMI 1640. The diluted blood wasplated at 60 μl/well in a 96-well plate and incubated for 15 minutes at37° C. Then 30 μl of serial dilutions in RPMI 1640 of the mouse IgG115C1, chimeric IgG1 15C1, mouse IgG1 15C1 containing human CH2 andchimeric IgG1 15C1 containing mouse CH2 MAbs were added to the blood andincubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

In the human whole blood assay, it was found that the recombinantversion containing the mouse CH2 domain had a similar inhibitoryactivity to that of the fully mouse IgG1 15C1 IgG1. On the other hand,the recombinant version containing the human CH2 was not as potent asthe fully mouse IgG1 15C1 IgG1, but had an activity reduced to that ofthe chimeric IgG1 15C1 (FIG. 5B). From these data, it was concluded thatthe Fc-mediated inhibitory contribution is restricted to the CH2 domainof the heavy chain of mouse IgG1.

Studies were then designed to evaluate which residues of the mouse CH2domain are necessary for Fc-mediated inhibitory activity. Starting withthe chimeric IgG115C1 containing the full length mouse CH2 domain, theapproach was to identify the critical region of the CH2 domain byintroducing a maximum number of human residues without observing a lossof the Fc-mediated inhibitory activity. The identification of thesecritical residues was determined by subdividing the mouse CH2 into 4parts (A: EU positions 231 to 261; B: EU positions 319 to 340; C: EUpositions 296-318 and D: EU positions 262 to 295) and introducing foreach of the 4 sub-regions the corresponding CH2 amino acid sequence of ahuman IgG1 (see FIG. 6A). The sub-division of the mouse CH2 into foursubregions was purely based on amino acid sequence homology with thehuman CH2. The four heavy chain mutants were engineered by overlappingPCR and recombinant MAbs expressed in PEAK. The corresponding antibodieswere purified from transfected-cell supernatants by protein G affinitycolumn chromatography and binding to CHO stable cell line expressinghuman TLR4-MD2 complex on their surface of the following antibodies wasdetermined: muCH2-chimeric IgG1 15C1; chimeric IgG1 15C1; A mu 231-261CH2 chimeric IgG1 15C1; B mu 319-340 CH2 chimeric IgG1 15C1; C mu296-318 CH2 chimeric IgG1 15C1; and D mu 262-295 CH2 chimeric IgG1 15C1.4×10⁵ cells/well were incubated for 30 minutes at 4° C. in 50 μl ofphosphate buffered saline (PBS) with 1% bovine serum albumin (PBS-1%BSA) and either serial dilution of the appropriate antibody or anirrelevant human IgG1 isotype control. Cells were washed once withPBS-1% BSA and incubated in the same buffer with FMAT-Blue®-conjugatedgoat anti-human Kappa light chain antibody (1:250 dilution, Sigma K3502)for 30 minutes at 4° C. Cells were washed twice with PBS-1% BSA andanalyzed using a FACScalibur® flow cytometer (Applied Biosystems) in theFL-4 channel.

Equivalent binding to TLR4/MD2 was demonstrated by FACS analysis (seeFIG. 6B). The neutralizing capability of these antibodies was thenevaluated using the human whole blood assay. Fresh heparinated bloodfrom healthy volunteers was obtained by venipuncture and diluted 1:2with RPMI 1640. The diluted blood was plated at 60 μl/well in a 96-wellplate and incubated for 15 minutes at 37° C. Then 30 μl of serialdilutions in RPMI 1640 of mutants chimeric IgG1 15C1 containingdifferent sub-regions of mouse CH2 MAbs were added to the blood andincubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

In a human whole blood assay (FIG. 6C), the inhibitory activity of theantibody containing the mouse CH2 domain was found to be drasticallyreduced when the human IgG1 amino acid residues at EU positions 319 to340 (mutant B) were introduced. It was concluded from this set ofmutants that the inhibitory activity within the mouse CH2 domain ispredominantly restricted to the amino acid sequence containing the mouseIgG1 residues at EU positions 319 to 340.

Studies were then designed to determine the effect of grafting of mouseIgG1 CH2 amino acid residues at positions 319 to 340 into the human CH2domain of the chimeric IgG1 15C1 antibody. The residues at EU positions319 to 340 of the mouse IgG1 CH2 were introduced within the CH2 of thechimeric IgG1 15C1 by overlapping PCR (see FIG. 7A). The mutant MAb wasexpressed in PEAK cells and purified from transfected-cell supernatantsby protein G affinity column chromatography. The neutralizing capabilityof chimeric IgG1 15C1, muCH2-chimeric IgG1 15C1 and mu319-340 chimericIgG1 15C1 antibodies was evaluated using the human whole blood assay.Fresh heparinated blood from healthy volunteers was obtained byvenipuncture and diluted 1:2 with RPMI 1640. The diluted blood wasplated at 60 μl/well in a 96-well plate and incubated for 15 minutes at37° C. Then 30 μl of serial dilutions in RPMI 1640 of the chimeric IgG115C1 and chimeric IgG1 15C1 containing either full length mouse CH2 ormouse CH2 residue 319 to 340 (EU numbering) MAbs were added to the bloodand incubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

In a human whole blood assay (FIG. 7B), the inhibitory activity of theantibody containing the mouse CH2 amino acid residues at EU positions319 to 340 was increased to a level similar to that of 15C1 antibodiescontaining the full length mouse CH2 domain. It was concluded thatgrafting into the human CH2 of a stretch of 22 mouse IgG1 amino acidresidues (EU positions 319 to 340) was sufficient to regain fullinhibitory activity to a level equivalent to that seen with the wholemouse IgG115C1.

Studies were then designed to determine the minimum number of residueswithin the mouse IgG1, EU positions 319 to 340, CH2 domain that arenecessary and sufficient to maintain the overall inhibitory activity ofthe mouse IgG1 Fc. From the alignment of the amino acid sequence ofamino acid residues at positions 319 to 340 between the mouse and humanIgG1, 7 differences were seen (numbered 1 to 7 in FIG. 8A). In order todetermine amongst these 7 residues those which are critical formaintaining the biological activity, the effect of exchanging theresidues of mouse IgG1 with those of human IgG1 was examined. To thisend, starting from chimeric IgG1 15C1 containing the mouse CH2 region EUposition 319 to 340, a set of 5 mutants were engineered using theQuickChange mutagenesis protocol from Stratagene (mutants A to E in FIG.8B). The 5 mutants MAbs were expressed in PEAK and purified fromtransfected-cell supernatants by protein G affinity columnchromatography.

The neutralizing capability of the 5 mutant MAbs was evaluated in thehuman whole blood assay. Fresh heparinated blood from healthy volunteerswas obtained by venipuncture and diluted 1:2 with RPMI 1640. The dilutedblood was plated at 60 μl/well in a 96-well plate and incubated for 15minutes at 37° C. Then 30 μl of serial dilutions in RPMI 1640 of thechimeric IgG1 15C1, chimeric IgG1 15C1 containing mouse CH2 amino acidresidues at EU positions 319 to 340 and mutants A to E MAbs were addedto the blood and incubated for an hour at 37° C. Blood cells were thenstimulated by adding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI1640 containing 0.1% HSA) to the wells and incubated for 6 hours. IL-6production was then measured by ELISA.

In a human whole blood assay (FIG. 8C), the mutants D and E exhibitedthe same inhibitory activity as chimeric IgG1 15C1 whereas mutants A, Band C showed an inhibitory activity at least as good as that of chimericIgG1 15C1 containing the mouse CH2 region EU positions 319 to 340. Itwas concluded from the results of the human whole blood assay and theanalysis of the amino acid sequences of the 5 mutants that on one hand,the introduction of the 4 human amino acids residues; Y1 (EU position319), K2 (EU position 322), S3 (EU position 324) and A7 (EU position339) (see FIG. 8B) had no deleterious effect on the inhibitory activityof the corresponding antibodies, but on the other hand the introductionof the 2 human amino acid residues N4 (EU position 325) and L6 (EUposition 326) had a negative effect with the loss of inhibitory effectdue to the Fc portion. Overall, it was concluded that out of the 7 mouseresidues potentially responsible for the inhibitory effect of the mouseIgG1 Fc, only three; Ser 4 (EU position325), Ala 5 (EU position 326) andPhe 6 (EU position 328); appeared to be critical for the overallinhibitory activity of the mouse IgG1 Fc.

Studies were then designed to determine the minimum number of mouseresidues which when grafted into the human CH2 domain of the chimericIgG1 15C1 antibody at the corresponding EU positions, would regain theinhibitory potency of the native mouse IgG1 15C1 antibody. Havingidentified within the mouse IgG1 CH2 domain, three amino acid residues ,Ser 326, Ala 327 and Phe 329 (EU numbering) as being responsible for theoverall inhibitory activity of mouse IgG1 15C1; a new set of mutantswere designed in order to introduce within the chimeric IgG1 15C1, all 6possible combinatorial combinations between mouse and human sequences atthese three EU positions. The sequences of the new mutants, F, G and H,together with the sequences of mutant C, D and E (described previouslyin FIG. 8B) are shown below in Table 1.

TABLE 1 Amino acid sequences of the 6 mutants (C to H) at EU positions325 to 328 within the CH2 domain of chimeric IgG1 15C1. Human residuesare in bold. mutations from number of amino acid human to mouse residuesat mouse amino acid residues left EU positions chimeric IgG1 residue atdefined overall in 325 to SEQ ID 15C1 mutants EU positions human CH2 328NO: human NKAL 97 # H L 328 F 1 NKAF 89 # E K 326 A 1 NAAL 98 # D N 325S; K 326 A 2 SAAL 99 # G K 326 A; L 328 F 2 NSAF 88 # F N 325 S; L 328 F2 SKAF 87 # C N 325 S; K 326 A 3 SAAF 86 Mouse SAAF 86

The 3 mutants were engineered using the Quick Change mutagenesisprotocol from Stratagene, expressed in PEAK cells and purified fromtransfected-cell supernatants by protein G affinity columnchromatography.

The relative binding affinity of these mutant antibodies was determinedusing a CHO stable cell line expressing human TLR4/MD2 on the cellsurface. 4×10⁵ cells/well were incubated for 30 minutes at 4° C. in 50μl of phosphate buffered saline (PBS) with 1% bovine serum albumin(PBS-1% BSA) and either serial dilution of the appropriate antibody oran irrelevant human IgG1 isotype control. Cells were washed once withPBS-1% BSA and incubated in the same buffer with FMAT-Blue®-conjugatedgoat anti-human Kappa light chain antibody (1:250 dilution, Sigma K3502)for 30 minutes at 4° C. Cells were washed twice with PBS-1% BSA andanalyzed using a FACScalibur® flow cytometer (Applied Biosystems) in theFL-4 channel.

The neutralizing capability of these mutated antibodies was alsoevaluated using the human whole blood assay. Fresh heparinated bloodfrom healthy volunteers was obtained by venipuncture and diluted 1:2with RPMI 1640. The diluted blood was plated at 60 μl/well in a 96-wellplate and incubated for 15 minutes at 37° C. Then 30 μl of serialdilutions in RPMI 1640 of the chimeric IgG1 15C1, chimeric IgG1 15C1containing mouse CH2 and mutant C, F and H MAbs were added to the bloodand incubated for an hour at 37° C. Blood cells were then stimulated byadding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI 1640 containing0.1% HSA) to the wells and incubated for 6 hours. IL-6 production wasthen measured by ELISA.

By FACS analysis, all the mutants were shown to have a similar relativeaffinity to the TLR4/MD2 complex expressed on CHO cells (see FIG. 9A) Ina human whole blood assay (FIG. 9B), the mutants C and F exhibited aninhibitory activity better than that of chimeric IgG1 15C1 containingthe mouse CH2, the mutant C being the more potent of the two. The mutantH was more potent than the chimeric IgG1 15C1 but less potent thanchimeric IgG1 15C1 containing the mouse CH2. Mutant D, E and G had aninhibitory activity similar but not better than that of the chimericIgG1 15C1.

In conclusion, two mutants within the CH2 domain of chimeric IgG1 15C1which have an inhibitory activity at least as good as that of the nativemouse IgG1 15C1 have been identified. These two mutants, C and F, haverespectively 3 and 2 amino acid residues mutated to the correspondingmouse residue of an IgG1 CH2 domain at the same EU position. Mutant Cwhich appears to have the strongest inhibitory activity of the twomutants has the following 3 mouse residues Ser, Ala and Phe at EUpositions 325, 326 and 328, respectively. Mutant F consists of the samemutations but only at EU positions 325 and 328.

Studies were then designed to evaluate whether the results obtained withchimeric IgG1 15C1 were also valid for the humanized version of 15C1.The humanized version of mouse mAb 15C1, consisting in the heavy chainversion 4-28 (15C1 Hu VH version 4-28) paired with the light chainversion A26 (15C1 Hu VL version A26) has been constructed byCDR-grafting as described herein and in International Patent applicationNo. PCT/IB2005/004174, the contents of which are hereby incorporated byreference in its entirety. This humanized version, later referred ashumanized IgG1 15C1, was previously shown by FACS analysis to have arelative affinity for the TLR4/MD2 complex expressed at the surface ofCHO cells similar to that of chimeric IgG1 15C1.

In a first set of experiments the inhibitory activity of the chimericIgG1 15C1 containing the mouse CH2 (muCH2 chim 15C1) was compared tothat of the humanized IgG1 15C1 with the mouse CH2 (muCH2 hum 15C1).These MAbs were expressed in PEAK cells and were purified fromtransfected-cell supernatants by protein G affinity columnchromatography. An equivalent binding to CHO-expressing TLR4/MD2 wasdemonstrated by FACS analysis.

The neutralizing capability of the muCH2 humanized 15C1 antibody wasevaluated using the human whole blood assay. Fresh heparinated bloodfrom healthy volunteers was obtained by venipuncture and diluted 1:2with RPMI 1640. The diluted blood was plated at 60 μl/well in a 96-wellplate and incubated for 15 minutes at 37° C. Then 30 μl of serialdilutions in RPMI 1640 of the mouse IgG1 15C1, chimeric IgG1 15C1, mouseIgG1 15C1 containing mouse CH2 and humanized IgG1 15C1 containing mouseCH2 MAbs were added to the blood and incubated for an hour at 37° C.Blood cells were then stimulated by adding 30 μl of E. coli K12 LPS (2ng/ml final in RPMI 1640 containing 0.1% HSA) to the wells and incubatedfor 6 hours. IL-6 production was then measured by ELISA.

In a human whole blood assay (FIG. 10), it was found that both thechimeric and humanized versions containing the mouse CH2 domain had asimilar inhibitory activity to that of the mouse IgG1 15C1 and that thisactivity was greater than seen with chimeric IgG1 15C1. It was concludedfrom these results that as previously seen for the chimeric IgG1 15C1,the inhibitory activity of the humanized IgG1 15C1 MAb can be increasedto a similar level as that of the native mouse IgG1 15C1 by theintroduction of the mouse CH2 domain in its Fc portion.

Similarly to the work done with chimeric IgG1 15C1, a set of 6 mutants Cto H (see Table 1) were constructed based on the use of either the humanor mouse amino acid residue at EU positions 325, 326 and 328 within theCH2 domain. The corresponding antibodies were purified fromtransfected-cell supernatants by protein G affinity columnchromatography. The binding affinity of these mutant antibodies wasdetermined using a CHO stable cell line expressing human TLR4/MD2 on thecell surface. 4×10⁵ cells/well were incubated for 30 minutes at 4° C. in50 μl of phosphate buffered saline (PBS) with 1% bovine serum albumin(PBS-1% BSA) and either serial dilution of the appropriate antibody oran irrelevant human IgG1 isotype control. Cells were washed once withPBS-1% BSA and incubated in the same buffer with FMAT-Blue®-conjugatedgoat anti-human Kappa light chain antibody (1:250 dilution, Sigma K3502)for 30 minutes at 4° C. Cells were washed twice with PBS-1%BSA andanalyzed using a FACScalibur® flow cytometer (Applied Biosystems) in theFL-4 channel.

By FACS analysis, all the mutants were shown to have a similar relativeaffinity to the TLR4/MD2 complex expressed on CHO cells (see FIG. 11A).

The neutralizing capability of the various mutant humanized antibodieswas evaluated using the human whole blood assay. Fresh heparinated bloodfrom healthy volunteers was obtained by venipuncture and diluted 1:2with RPMI 1640. The diluted blood was plated at 60 μl/well in a 96-wellplate and incubated for 15 minutes at 37° C. Then 30 μl of serialdilutions in RPMI 1640 of the humanized IgG1 15C1 mutants C, F, G and Hand humanized IgG1 15C1 containing the mouse CH2 MAbs were added to theblood and incubated for an hour at 37° C. Blood cells were thenstimulated by adding 30 μl of E. coli K12 LPS (2 ng/ml final in RPMI1640 containing 0.1% HSA) to the wells and incubated for 6 hours. IL-6production was then measured by ELISA.

In a human whole blood assay the inhibitory activity of the 6 mutants ascompared with that of muCH2 humanized IgG115C1. The results presented inFIG. 11B clearly show that mutant C and F are at least as potent asmuCH2 humanized IgG1 15C1 whereas mutants G and H are less efficient.Mutants D and E were also shown to be less efficient than muCH2humanized IgG1 15C1. The results concord well with those obtainedearlier for chimeric IgG1 15C1 (FIG. 9B).

Finally, the inhibitory activity of the best humanized CH2 mutant,mutant #C, containing three mouse amino acid residues at EU positions325 (Ser), 326 (Ala) and 328 (Phe) was tested in a human whole bloodassay along with the chimeric IgG1 15C1 and the humanized IgG1 15C1containing the mouse CH2 domain (muCH2 humanized 15C1). Freshheparinated blood from healthy volunteers was obtained by venipunctureand diluted 1:2 with RPMI 1640. The diluted blood was plated at 60μl/well in a 96-well plate and incubated for 15 minutes at 37° C. Then30 μl of serial dilutions in RPMI 1640 of chimeric IgG1 15C1, humanizedIgG1 15C1 mutant #C and humanized 15C1 containing mouse CH2 MAbs wereadded to the blood and incubated for an hour at 37° C. Blood cells werethen stimulated by adding 30 μl of E. coli K12 LPS (2 ng/ml final inRPMI 1640 containing 0.1% HSA) to the wells and incubated for 6 hours.IL-6 production was then measured by ELISA. The data presented in FIG.12 show that mutant #C has an inhibitory activity similar to that ofmuCH2 humanized IgG1 15C1 and much more potent than that of chimericIgG1 15C1.

The implication of CD32 in the increase of the inhibitory effect ofanti-TLR2, anti-MD2 and anti-CD14 mouse IgG1 monoclonal antibodies asmonitored by the inhibition of the pro-inflammatory cytokine IL-6 wasevaluated using the human whole blood assay. Fresh heparinated bloodfrom healthy volunteers was obtained by venipuncture and diluted 1:2with RPMI 1640. The diluted blood was plated at 60 μl/well in a 96-wellplate and incubated for 15 minutes at 37° C. Then 30 μl of serialdilutions in RPMI 1640 of mouse anti-TLR2 (13A), mouse anti-MD2 (18H10,13B) and mouse anti-CD14(13C) MAs with or without mouse anti-human CD32monoclonal antibody (Clone AT10, Catalog number 2125-3210, AbD Serotec)were added to the blood and incubated for an hour at 37° C. Blood cellswere then stimulated by adding 30 μl of E. coli K12 LPS (2 ng/ml finalin RPMI 1640 containing 0.1% HSA) to the wells and incubated for 6hours. IL-6 production was then measured by ELISA.

The results shown in FIGS. 13A-13C tend to demonstrate the involvementof interactions between the MAb Fc portion and human FcγRIIA in aputative inhibitory response in other systems than the TLR4 i.e, TLR2,MD2 and CD14.

The alignment of the CH2 domain of all human, mouse and rat IgG isotypesin FIG. 16, shows that apart from mouse IgG 1, rat IgG2a also containsan SAAF motif (SEQ ID NO: 86) at EU positions 325-328 whereas rat IgG 1contains a very homologous SGAF (SEQ ID NO: 100) sequence at the same EUpositions. None of the other human, mouse or rat IgG isotypes containthis SAAF motif (SEQ ID NO: 86). The EU numbering (Edelman, G. M. etal., 1969, Proc. Natl Acad. Sci. USA 63, 78-85) for the gamma chains ofthe CH2 domain starts at 231 and ends at 340. The human IgG1, IgG3 andIgG4, the mouse IgG2ab, IgG2aa, IgG2b and IgG3, and the rat IgG2b CH2exons encode 110 amino acids. The human IgG2 and rat IgG2c CH2 exonencode 109 amino acids due to a three nucleotide (nt) deletion. Themouse IgG1, rat IgG 1 and IgG2a CH2 exon encode 107 amino acids due to anine nt deletion.

Example 4 CDR3 Mutated Neutralizing Antibodies

The studies described herein are directed methods of increasing thepotency of neutralizing antibodies by modifying one or more residues inthe CDR3 portion of an antibody. In particular, the studies describedherein use an altered neutralizing antibody that recognizes the TLR4/MD2complex. These anti-TLR4/MD2 antibodies are modified to include one ormore mutations in the CDR3 portion. These antibodies include thefollowing sequences, wherein the single point mutation with the CDR3region has been underlined within the amino acid sequence:

15C1 humanized VH mutant 1 amino acid sequence: (SEQ ID NO: 66)QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKD PSDAFPYWGQGTLVTVSS15C1 humanized VH mutant 1 nucleic acid sequence: (SEQ ID NO: 67)CAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGACGCCTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TTCC15C1 humanized VH mutant 2 amino acid sequence: (SEQ ID NO: 68)QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKD PSEGFPYWGQGTLVTVSS15C1 humanized VH mutant 2 nucleic acid sequence: (SEQ ID NO: 69)CAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGAGGGATTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TTCCAs compared to the reference humanized 4-28 15C1 VH sequence:

15C1 humanized VH 4-28 amino acid sequence: (SEQ ID NO: 45)QVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPGKGLEWMGYIHYSGYTDFNPSLKTRITISRDTSKNQFSLKLSSVTAVDTAVYYCARKD PSDGFPYWGQGTLVTVSS15C1 humanized VH 4-28 nucleic acid sequence: (SEQ ID NO: 70)CAGGTGCAGCTTCAGGAGTCCGGCCCAGGACTGGTGAAGCCTTCGGACACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATCACCGGTGGTTATAGCTGGCACTGGATACGGCAGCCCCCAGGGAAGGGACTGGAGTGGATGGGGTATATCCACTACAGTGGTTACACTGACTTCAACCCCTCCCTCAAGACTCGAATCACCATATCACGTGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGTGGACACTGCAGTGTATTACTGTGCGAGAAAAGATCCGTCCGACGGATTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TTCC15C1 humanized VL mutant 1 amino acid sequence: (SEQ ID NO: 71)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNSHSFPLTFGG GTKVEIK15C1 humanized VL mutant 1 nucleic acid sequence: (SEQ ID NO: 72)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGAATAGTCACAGTTTTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA15C1 humanized VL mutant 2 amino acid sequence: (SEQ ID NO: 73)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQGHSFPLTFGG GTKVEIK15C1 humanized VL mutant 2 nucleic acid sequence: (SEQ ID NO: 74)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGCAGGGTCACAGTTTTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA15C1 humanized VL mutant 3 amino acid sequence: (SEQ ID NO: 75)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNSSSFPLTFGG GTKVEIK15C1 humanized VL mutant 3 nucleic acid sequence: (SEQ ID NO: 76)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGAATAGTAGTAGTTTTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA15C1 humanized VL mutant 4 amino acid sequence: (SEQ ID NO: 77)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSHSFPLTFGG GTKVEIK15C1 humanized VL mutant 4 nucleic acid sequence: (SEQ ID NO: 78)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGCAGAGTCACAGTTTTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAAAs compared to the reference 15C1 humanized VL A26:

15C1 humanized VL A26 amino acid sequence: (SEQ ID NO 48)EIVLTQSPDFQSVTPKEKVTITCRASQSISDHLHWYQQKPDQSPKLLIKYASHAISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQNGHSFPLTFGG GTKVEIK15C1 humanized VL A26 nucleic acid sequence: (SEQ ID NO: 79)GAAATTGTGTTGACGCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAAAAAGTCACCATCACCTGCAGGGCCAGTCAGAGTATCAGCGACCACTTACACTGGTACCAACAGAAACCTGATCAGTCTCCCAAGCTCCTCATCAAATATGCTTCCCATGCCATTTCTGGGGTCCCATCGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAATAGCCTAGAGGCTGAAGATGCTGCAACGTATTACTGTCAGAATGGTCACAGTTTTCCGCTCACTTTCGGCGGA GGGACCAAGGTGGAGATCAAA

The binding of these mutants was analyzed by F ACS on cells expressingrecombinant human TLR4-MD2. PEAK cells were co-transfected with 1 ug ofcombinations of expression vectors encoding 15C1 humanized VH mutant 1or 2 together with 15C1 humanized VL A26 or 15C1 humanized VH 4-28together with 15C1 humanized VL mutant 1, 2, 3 or 4 shown in Example 4using the Trans IT-LT 1 transfection reagent (Minis Bio Corporation,Madison Wis.). All transfections were carried out in duplicates. 72 hpost-transfection, PEAK cells supernatants were collected and theconcentration of recombinant human IgG I/Kappa measured by ELISA. Theantibody concentrations of all the supernatants were then adjusted to0.33 μg/ml. These supernatants and two serial dilutions of 0.11 and 0.04ug/ml were then tested for binding to CHO cells expressing humanTLR4-MD2 at their surface by FACS. 5×10⁵ cells were incubated with thediluted PEAK supernatant for 1 hat 4° C. Following two washes, cellswere incubated with secondary antibody (allophycocyanin-conjugated goatanti-human IgG antibody (1:200 dilution; Molecular Probes). Cells wereanalyzed using a FACSCalibur flow cytometer (BD Biosciences) in the FL-4channel (FIGS. 17A-17G). The binding of the humanized mutant versions of15C1 to TLR4 in FIGS. 17A-17G is expressed as the mean fluorescenceintensity for the different antibody concentrations. One representativeexperiment of four. Error bars show±S.D. The version VH mutant 1 and VLmutant 2 appear to have a higher MFI than the patented humanized versionindicating that these two versions have a higher relative affinity thanthe humanized version.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An altered polypeptide comprising at least anFcγR binding portion of an Fc region wherein the polypeptide comprisesat least one mutation compared to a starting polypeptide and wherein atleast one mutation is selected from a group consisting of a substitutionat EU amino acid positions 325, 326 and
 328. 2. The altered polypeptideof claim 1, wherein the altered polypeptide elicits a modified Fc gammareceptor activity.
 3. The altered polypeptide of claim 1, wherein thealtered polypeptide is an antibody or fragment thereof or a fusionprotein.
 4. The altered polypeptide of claim 1, wherein the FcγR bindingportion or the Fc region is derived from a mouse, rat or human antibody.5. The altered polypeptide of claim 4, wherein the FcγR binding portioncomprises a complete Fc region.
 6. The altered polypeptide of claim 2,wherein the modified Fc gamma receptor activity is the inhibition of therelease of proinflammatory mediators.
 7. The altered polypeptide ofclaim 2, wherein said Fc gamma receptor is the human CD32A.
 8. Thealtered polypeptide of claim 3, wherein said antibody is human IgG1isotype, human IgG2 isotype, human IgG3 isotype or human IgG4 isotype.9. The altered polypeptide of claim 8, comprising a mutation selectedfrom the group consisting of the amino acid residue at EU position 325is substituted with serine; the amino acid residue at EU position 326 issubstituted with alanine; the amino acid residue at EU position 328 issubstituted with phenylalanine; and combinations thereof.
 10. Thealtered polypeptide of claim 9, wherein said heavy chain constant regioncomprises two or more amino acid substitutions with an amino acidresidue that is different from the corresponding amino acid residue inan unaltered antibody, wherein said substitutions occur at two or moreamino acid residues selected from EU positions 325, 326 and 328 of saidheavy chain constant region.
 11. The altered polypeptide of claim 10,wherein EU position 326 of the heavy chain constant region issubstituted with alanine and EU position 328 of the heavy chain constantregion is substituted with phenylalanine.
 12. The altered polypeptide ofclaim 9, wherein EU positions 325-328 of the heavy chain constant regionconsist of a sequence selected from SAAF (SEQ ID NO: 86), SKAF (SEQ IDNO: 87) and NKAF (SEQ ID NO: 89).
 13. The altered polypeptide of claim1, wherein said polypeptide binds to a target selected from a toll-likereceptor (TLR), MD2 accessory protein, soluble TLR4, the TLR4/MD2complex, both soluble TLR4 and the TLR4/MD2 complex, TLR2, and CD14. 14.A method of activating ICAM signaling, said method comprising bindinghuman FcγR CD32A with an altered polypeptide comprising at least an FcγRbinding portion of an Fc region wherein the polypeptide comprises atleast one mutated amino acid residue of the heavy chain constant regionselected from one or more of the amino acid residues that correspond toEU positions 325, 326 and 328 with an amino acid residue that isdifferent from the corresponding amino acid residue in an unalteredpolypeptide, wherein said altered polypeptide elicits an inhibition ofproinflammatory mediators release by ligation with human FcγR CD32Awhile retaining binding to antigen via its Fv region as compared to anunaltered polypeptide.
 15. The method of claim 14, wherein said alteredpolypeptide is an altered antibody.
 16. The method of claim 15, whereinthe altered antibody comprises a mutation selected from the groupconsisting of the amino acid residue at EU position 325 is substitutedwith serine; the amino acid residue at EU position 326 is substitutedwith alanine; the amino acid residue at EU position 328 is substitutedwith phenylalanine; and combinations thereof.
 17. The method of claim15, wherein said heavy chain constant region of said altered antibodycontains two or more substitutions, with an amino acid residue that isdifferent from the corresponding amino acid residue in an unalteredantibody, wherein said substitutions occur at two or more amino acidresidues selected from residues that correspond to EU positions 325, 326and
 328. 18. The method of claim 15, wherein the residue at EU position326 of said altered antibody is substituted with alanine and the residueat EU position 328 of said altered antibody is substituted withphenylalanine.
 19. The method of claim 15, wherein residues at EUpositions 325-328 of said altered antibody consist of a sequenceselected from SAAF (SEQ ID NO: 86), SKAF (SEQ ID NO: 87) and NKAF (SEQID NO: 89).
 20. The method of claim 15, wherein said altered antibody toa target selected from a toll-like receptor (TLR), MD2 accessoryprotein, soluble TLR4, the TLR4/MD2 complex, both soluble TLR4 and theTLR4/MD2 complex, TLR2, and CD14.
 21. An isolated polypeptide comprisinga gamma Fc (γFc) region, wherein residues at EU positions 325-328 ofsaid region consist of an amino acid motif selected from SAAF (SEQ IDNO: 86) and NKAF (SEQ ID NO: 89).